Showing papers on "Oligonucleotide published in 1985"
TL;DR: The high efficiency, approximately equal to 10-fold greater than that observed using current methods without enrichment procedures, is obtained by using a DNA template containing several uracil residues in place of thymine, which is applied to mutations introduced via both oligonucleotides and error-prone polymerization.
Abstract: Several single-base substitution mutations have been introduced into the lacZ alpha gene in cloning vector M13mp2, at 40-60% efficiency, in a rapid procedure requiring only transfection of the unfractionated products of standard in vitro mutagenesis reactions. Two simple additional treatments of the DNA, before transfection, produce a site-specific mutation frequency approaching 100%. The approach is applicable to phenotypically silent mutations in addition to those that can be selected. The high efficiency, approximately equal to 10-fold greater than that observed using current methods without enrichment procedures, is obtained by using a DNA template containing several uracil residues in place of thymine. This template has normal coding potential for the in vitro reactions typical of site-directed mutagenesis protocols but is not biologically active upon transfection into a wild-type (i.e., ung+) Escherichia coli host cell. Expression of the desired change, present in the newly synthesized non-uracil-containing covalently closed circular complementary strand, is thus strongly favored. The procedure has been applied to mutations introduced via both oligonucleotides and error-prone polymerization. In addition to its utility in changing DNA sequences, this approach can potentially be used to examine the biological consequences of specific lesions placed at defined positions within a gene.
8,474 citations
TL;DR: A clone encoding PrP 27-30, the major protein in purified preparations of scrapie agent, was selected from a scrapie-infected hamster brain cDNA library by oligonucleotide probes corresponding to the N terminus of the protein.
1,514 citations
TL;DR: The human erythropoietin gene has been isolated from a genomic phage library by using mixed 20-mer and 17-mer oligonucleotide probes and encodes a 27-amino acid signal peptide and a 166-AMino acid mature protein with a calculated Mr of 18,399.
Abstract: The human erythropoietin gene has been isolated from a genomic phage library by using mixed 20-mer and 17-mer oligonucleotide probes. The entire coding region of the gene is contained in a 5.4-kilobase HindIII-BamHI fragment. The gene contains four intervening sequences (1562 base pairs) and five exons (582 base pairs). It encodes a 27-amino acid signal peptide and a 166-amino acid mature protein with a calculated Mr of 18,399. The erythropoietin gene, when introduced into Chinese hamster ovary cells, produces erythropoietin that is biologically active in vitro and in vivo.
1,122 citations
TL;DR: Two methods are described by which this approach has been used to produce mutations in M13mp2 phage DNA with high efficiency as a result of simple and rapid in vitro manipulations.
Abstract: M13 RF IV DNA may be prepared in vitro to contain phosphorothioate-modified internucleotidic linkages in the (-)strand only. Certain restriction enzymes react with this modified DNA to hydrolyze the (+)strand exclusively when a phosphorothioate linkage occurs at the normal cleavage point in the (-)strand. The reaction of Pvu I with M13mp2 RF IV DNA containing dCMPS residues in the (-)strand is of this type, and is exploited to allow subsequent digestion with exonuclease III of a portion of the (+)strand opposite different mutagenic mismatched oligonucleotide primers. Two methods are described by which this approach has been used to produce mutations in M13mp2 phage DNA with high efficiency as a result of simple and rapid in vitro manipulations. Plaques containing mutant phage in a genetically-pure form are obtained at a frequency of 40-66%, allowing their characterisation directly by sequence analysis without prior screening and plaque purification. The wide applicability of this approach is discussed.
938 citations
TL;DR: An improved method is described for the construction of mutations in M13 vectors using synthetic oligonucleotides with multiple mismatches to the template, which offers an alternative strategy for construction of multiple mutations over a short region.
Abstract: An improved method is described for the construction of mutations in M13 vectors using synthetic oligonucleotides. The DNA is first cloned into a novel M13 vector (based upon M13mp18 or M13mp19), which carries a genetic marker that can be selected against, such as an EcoK or EcoB site, or an amber mutation in an essential phage gene. In this "coupled priming" technique, one primer is used to construct the silent mutation of interest, and a second primer is used to eliminate the selectable marker on the minus strand. After primer extension and ligation, the heteroduplex DNA is transfected into a strain of E. coli which is repair deficient and selects against the plus strand marker. Over 50 mutants have been constructed with this approach, and the yields can be excellent (up to 70%). For the stepwise construction of mutations using separate rounds of mutagenesis, the EcoK and EcoB markers offer a particular advantage over the amber marker. They permit selection in each round, as it is possible to cycle between the two markers. However for construction of multiple mutations over a short region, long synthetic oligonucleotides with multiple mismatches to the template can offer an alternative strategy.
818 citations
TL;DR: An oligonucleotide hybridization procedure has been developed that eliminates the preferential melting of A X T versus G X C base pairs, allowing the stringency of the hybridization to be controlled as a function of probe length only.
Abstract: An oligonucleotide hybridization procedure has been developed that eliminates the preferential melting of A X T versus G X C base pairs, allowing the stringency of the hybridization to be controlled as a function of probe length only. This technique, which uses tetramethylammonium chloride, is especially helpful whenever a highly complex library is screened with a pool of oligonucleotide probes, which usually vary widely in base composition. The procedure can also be applied advantageously whenever an exact match to an oligonucleotide probe is desired, such as in screening for clones having as little as a single-base alteration generated by in vitro mutagenesis.
798 citations
TL;DR: Results demonstrate that the clones isolated correspond to the ER mRNA sequence, and use of lambda OR8 as a hybridization probe revealed a single poly(A)+ RNA band of approximately equal to 6.2 kilobase pairs in the ER-containing human breast cancer cell lines MCF-7 and T47D.
Abstract: Poly(A)+ RNA isolated from the human breast cancer cell line MCF-7 was fractionated by sucrose gradient centrifugation and fractions enriched in estrogen receptor (ER) mRNA were used to prepare randomly primed cDNA libraries in the lambda gt10 and lambda gt11 vectors. Clones corresponding to ER sequence were isolated from both libraries after screening with either ER monoclonal antibodies (lambda gt11) or synthetic oligonucleotide probes designed from two peptide sequences of purified ER (lambda gt10). Five cDNA clones were isolated by antibody screening and five were isolated after screening with synthetic oligonucleotides. The two largest ER cDNA clones, lambda OR3 (1.3 kilobase pairs) and lambda OR8 (2.1 kilobase pairs), isolated by using antibodies and oligonucleotides, respectively, were able to enrich selectively for ER mRNA by hybrid-selection. Furthermore, lambda OR8 contains the DNA sequence expected from the two ER peptides and crosshybridizes with each of the other ER cDNA clones. These results demonstrate that the clones isolated correspond to the ER mRNA sequence. Use of lambda OR8 as a hybridization probe revealed a single poly(A)+ RNA band of approximately equal to 6.2 kilobase pairs in the ER-containing human breast cancer cell lines MCF-7 and T47D. In contrast, no hybridization was seen in the human ER-negative cell line HeLa. The same probe hybridizes to a chicken gene that is expressed in oviduct tissue as a 7.5-kilobase-pair poly(A)+ RNA.
704 citations
TL;DR: Oligonucleotide probes with deoxyinosine residues at ambiguous points seem to be useful as hybridization probes for cloning genes for proteins containing amino acids with degenerate codons.
507 citations
TL;DR: A rapid and versatile method has been developed for the synthesis of oligonucleotides which contain an aliphatic amino group at their 5' terminus that reacts specifically with a variety of electrophiles, thereby allowing other chemical species to be attached to the oligon nucleotide.
Abstract: A rapid and versatile method has been developed for the synthesis of oligonucleotides which contain an aliphatic amino group at their 5' terminus. This amino group reacts specifically with a variety of electrophiles, thereby allowing other chemical species to be attached to the oligonucleotide. This chemistry has been utilized to synthesize several fluorescent derivatives of an oligonucleotide primer used in DNA sequence analysis by the dideoxy (enzymatic) method. The modified primers are highly fluorescent and retain their ability to specifically prime DNA synthesis. The use of these fluorescent primers in DNA sequence analysis will enable DNA sequence analysis to be automated.
408 citations
TL;DR: Electrophoretic separation of DNA heteroduplexes in a well-characterized gel system is described, showing that four different human β-thalassaemia alleles with known single base mutations can be detected with as little as 5 µg of total genomic DNA.
Abstract: Certain single base substitutions causing genetic diseases or resulting in polymorphisms linked to mutant alleles, alter a restriction enzyme cleavage site and can therefore be detected in total genomic DNA using DNA blots. Many base substitutions do not lead to an altered restriction site, but these can be detected using synthetic oligonucleotides as hybridization probes if the DNA sequence surrounding the base substitution is known. In the case of beta-thalassaemia, where 22 different single base mutations have been identified, a large number of probes would be required for diagnosis. An approach which was used to detect mutations in viral DNA involves the S1 nuclease treatment of heteroduplexes formed between wild-type and mutant DNA. Although certain single base mismatches are cleaved by S1 nuclease (ref. 11 and T. Shenk, personal communication), many other mismatches examined by this procedure are not cleaved (B. Seed, personal communication; R.M.M., unpublished data). Heteroduplexes between mutant and wild-type subgenomic fragments of double-stranded reovirus RNA migrate slower than the corresponding homoduplexes in polyacrylamide gels containing 7 M urea, but it is not known whether this method is applicable to DNA heteroduplexes containing single base mismatches. Here we describe a procedure that involves the electrophoretic separation of DNA heteroduplexes in a well-characterized gel system. We show that four different human beta-thalassaemia alleles with known single base mutations can be detected with as little as 5 micrograms of total genomic DNA. The method should be useful in the localization and diagnosis of mutations associated with genetic diseases.
403 citations
TL;DR: Results support the notion that there is a common binding site for DNA on white blood cells, that the stoichiometry of the association is compatible with a ligand receptor relationship, and that this apparent receptor is responsible for the endocytosis and degradation of exogenous DNA.
Abstract: Previous studies have indicated that white blood cells possess DNA on their outer membranes. In this study we set out to determine whether exogenous DNA bound to cells in a fashion compatible with a ligand receptor union. Purified populations of white blood cells; neutrophils (polymorphonuclear leukocytes, PMN), adherent mononuclear cells (ADMC), rosetting lymphocytes (E+ cells), and nonrosetting lymphocytes (E- cells) were incubated with radiolabeled lambda phage DNA in increasing concentrations. Binding of [3H]DNA was a saturable process and was inhibited by excess cold DNA and prior trypsinization of the cells. Rate zonal density centrifugation of purified cell membrane preparations confirmed that DNA was binding to the outer cell surface. The dissociation constant for all four cell types was approximately 10(-9) M, and from 0.81 X 10(3) to 2.6 X 10(3) molecules of lambda phage DNA bound to each cell depending upon cell type. Binding was not competitively inhibited by RNA, polydeoxyadenylic acid-polydeoxythymidylic acid (poly [d(A).d(T)]), or mononucleotides. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE)-separated proteins from PMN, ADMC, E+, and E- cells were electrophoretically blotted onto nitrocellulose sheets; a probe of biotin-labeled DNA indicated a single species of DNA-binding molecule migrating in a position consistent with a molecular weight of 30,000. Isotopic and immunofluorescent studies indicate that DNA is internalized and degraded to oligonucleotides; this process is inhibited by cycloheximide. These results support the notion that there is a common binding site for DNA on white blood cells, that the stoichiometry of the association is compatible with a ligand receptor relationship, and that this apparent receptor is responsible for the endocytosis and degradation of exogenous DNA.
TL;DR: A rapid and efficient microscale method for in vitro site-directed mutagenesis by gene synthesis and its potential for the extensive mutational analysis of genes is described.
Abstract: We describe a rapid and efficient microscale method for in vitro site-directed mutagenesis by gene synthesis. Mutants are constructed by "shot-gun ligation" of overlapping synthetic oligonucleotides yielding double stranded synthetic DNA of more than 120 nucleotides in length. The terminal oligonucleotides of the DNA segment to be synthesized are designed to create sticky ends complementary to unique restriction sites of a polylinker present in an M13 vector. The oligonucleotides are hybridized and ligated to the M13 vector without any purification of the synthetic DNA segment. After cloning, about half of the progeny from such shot-gun ligations contained the predicted sequence demonstrating the efficacy of this method for gene synthesis and its potential for the extensive mutational analysis of genes.
TL;DR: Oligonucleotides containing a free sulphydryl group at their 5'-termini have been synthesised and further derivatised with thiol specific probes, but are unstable at pH 8 decomposing to the free 5'-phosphate and so are unsuitable for further deriv atisation.
Abstract: Oligonucleotides containing a free sulphydryl group at their 5'-termini have been synthesised and further derlvatised with thiol specific probes. The nucleotide sequence required is prepared using standard solid phase phosphoramidlte techniques and an extra round of synthesis is then performed using the S-trlphenylmethyl O-methoxymorpholinophosphite derivatives of 2-mercaptoethanol, 3-mercaptopropan (T)ol or 6-mercaptohexan(l)ol. After cleavage from the resin and removal of the phosphate and base protecting groups, this yields an oligonucleotide containing an S-triphenylmethyl group attached to the 5'-phosphate group via a two, three or six carbon chain. The triphenylmethyl group can be readily removed with silver nitrate to give the free thiol. With the three and six carbon chain oligonucleotides, this thiol can be used, at pH 8, for the attachment of thiol specific probes as illustrated by the reaction with fluorescent conjugates of iodoacetates and maleiimides. However, oligonucleotides containing a thiol attached to the 5'-phosphate group via a two carbon chain are unstable at pH 8 decomposing to the free 5'phosphate and so are unsuitable for further derivatisation.
TL;DR: Addition of DNA polymerase I and helicase II (UvrD protein) to the reaction mixture stimulates the turnover rate of the excision nuclease to a level comparable to that observed in vivo.
Abstract: UvrABC excision nuclease (UvrA, UvrB, and UvrC proteins) of Escherichia coli removes nucleotide mono- and diadducts from DNA in the form of oligonucleotides 12 or 13 bases long. We find that the purified enzyme dissociates from DNA very slowly, if at all, in the absence of other proteins implicated in excision repair. Addition of DNA polymerase I and helicase II (UvrD protein) to the reaction mixture stimulates the turnover rate of the excision nuclease to a level comparable to that observed in vivo.
TL;DR: It is shown that release of the damaged fragment from the parental uvrABC-incised DNA is dependent upon either chelating conditions or the simultaneous addition of the uvrD gene product and the polA gene product when polymerization of deoxynucleoside triphosphates is concomitantly catalyzed.
Abstract: The bimodal-incision nature of the reaction of UV-irradiated DNA catalyzed by the Escherichia coli uvrABC protein complex potentially leads to excision of a 12- to 13-nucleotide-long damaged fragment However, the oligonucleotide fragment containing the UV-induced pyrimidine dimer is not released under nondenaturing in vitro reaction conditions Also, the uvrABC proteins are stably bound to the incised DNA and do not turn over after the incision event In this communication it is shown that release of the damaged fragment from the parental uvrABC-incised DNA is dependent upon either chelating conditions or the simultaneous addition of the uvrD gene product (helicase II) and the polA gene product (DNA polymerase I) when polymerization of deoxynucleoside triphosphate substrates is concomitantly catalyzed The product of this multiprotein-catalyzed series of reactions serves as a substrate for polynucleotide ligase, resulting in the restoration of the integrity of the strands of DNA The addition of the uvrD protein to the incised DNA-uvrABC complex also results in turnover of the uvrC protein It is suggested that the repair processes of incision, excision, resynthesis, and ligation are coordinately catalyzed by a complex of proteins in a "repairosome" configuration
TL;DR: DNase I-protection mapping patterns reveal binding to two 30-base-pair regions at the boundaries of the sequence of GAL1-GAL10 promoter, likely to be the GAL4 gene product.
Abstract: A binding activity specific for the upstream activating sequence of the GAL1-GAL10 promoter of Saccharomyces cerevisiae has been purified 220-fold on the basis of a nitrocellulose filter-binding assay. The binding activity is enriched in a nuclear preparation and is likely to be the GAL4 gene product. DNase I-protection mapping patterns reveal binding to two 30-base-pair regions at the boundaries of the sequence. A nearly identical mapping pattern is obtained with the coordinately regulated GAL7 promoter. The four 30-base-pair regions of binding in the two promoters are closely homologous, with a core consensus sequence of C-G-CG-TG-C-A-A-C-A-G-T-G-C-T-C-C-G-A-A- GC-G-A-T. A synthetic oligonucleotide with such a sequence competes with the upstream activating sequence in the binding reaction.
TL;DR: Evidence is provided that antigenic variation in B. hermsii is associated with DNA rearrangements and with occurrence of expression-linked copies of all, or part, of an antigen-specifying gene.
TL;DR: A number of considerations in oligonucleotide probe design are described, including unique probe design from cDNA sequences and mixed Probe design from protein primary structure data, and the special considerations of specificity controls are addressed.
TL;DR: NC1, the C-terminal non-collagenous globular domain of collagen IV, represents one of the two end regions responsible for the assembly and cross-linking of the extracellular network of basement membrane collagen.
Abstract: NC1, the C-terminal non-collagenous globular domain of collagen IV, represents one of the two end regions responsible for the assembly and cross-linking of the extracellular network of basement membrane collagen. Several cDNA clones for the NC1 domain of the α1(IV) collagen chain of mouse have been isolated by using synthetic oligonucleotides as screening probes for mouse libraries. The oligonucleotides were synthesized according to known stretches of the corresponding protein sequence. Sequencing of the overlapping cDNA clones allowed the complete amino acid sequence of the NC1 domain to be deduced as well as the C-terminal 165 amino acid residues of the triple helix. It consists of 229 amino acid residues which comprise two homologous regions with a high content of cysteine. These DNA and protein sequences are compared to the corresponding sequences of other collagens and discussed with respect to their structural and biological significance.
TL;DR: By adding 50 35S-labeled nucleotides to the 3' end, the authors prepared a stable oligonucleotide probe with a sensitivity per unit of exposure time comparable to that of the unstable 5'-32P- labeled oligon nucleotide probe.
TL;DR: 5'-biotin-labeled oligonucleotides were hybridized to dot, Southern and genomic blots of target plasmid DNA immobilized on nitrocellulose filters and there is no noticeable disturbance of the strength and selectivity of hybridized probes in comparison with non-modified DNA.
Abstract: Oligodeoxynucleotides have been selectively labeled with biotin at their 5'-termini through an aminoalkylphosphoramide linker arm by an efficient chemical method. The reactions were performed in aqueous solution on unprotected oligonucleotides and were insensitive of the sequence and length of the oligonucleotide. 5'-biotin-labeled oligonucleotides were hybridized to dot, Southern and genomic blots of target plasmid DNA immobilized on nitrocellulose filters. Detection level is about 2 fmole. There is no noticeable disturbance of the strength and selectivity of hybridization of the 5'-biotin-labeled probes in comparison with non-modified DNA.
TL;DR: Radiolabeled oligonucleotide cDNA probes, synthesized by chemical and enzymatic means, provide strong hybridization in zones known to contain vasopressin cell bodies, and can provide insight into regional mRNA dynamics and may reflect functional activity of peptidergic neurons.
Abstract: Hybridizable vasopressin mRNA may be quantitatively localized in situ in sections from rat hypothalamus. Radiolabeled oligonucleotide cDNA probes, synthesized by chemical and enzymatic means, provide strong hybridization in zones known to contain vasopressin cell bodies. Multiple single-stranded 32P-, 35S-, or 3H-labeled oligonucleotides demonstrate localized hybridization that increases as probes are lengthened from 8 to 75 bases. Competition studies, RNase experiments, anatomic localization, and use of multiple probes all support hybridization specificity. An approximate doubling of hybridizable mRNA in both supraoptic and paraventricular nuclei can be detected with dehydration of the animals. Hybridizable mRNA densities are virtually normal in hypothalamic nuclei of Brattleboro rats given free access to water. These methods can provide insight into regional mRNA dynamics and may reflect functional activity of peptidergic neurons.
TL;DR: RNA sequences encoding the surface projection (spike) of the coronavirus infectious bronchitis virus, strain Beaudette, have been cloned into pBR322 using cDNA primed with a specific oligonucleotide.
Abstract: RNA sequences encoding the surface projection (spike) of the coronavirus infectious bronchitis virus, strain Beaudette, have been cloned into pBR322 using cDNA primed with a specific oligonucleotide. A 5.3 kilobase viral insert in the clone pMB179 has been identified. The region of this clone coding for the spike gene has been sequenced by the chain termination method, and we present here the first report of DNA sequence data for a coronavirus spike protein, the protein which forms the characteristic 'corona' after which the group is named. The amino acid sequence of the primary translation product, deduced from the DNA sequence, predicts a polypeptide of 1162 amino acids with a molecular weight of 127 006. This has many interesting features which confirm and extend our knowledge of this recently characterized membrane glycoprotein. The polypeptide is subsequently cleaved to S1 and S2, and partial amino acid analysis of the amino-terminus of the S1 polypeptide has been employed to locate the position of this terminus of S1 within the large open reading frame. The amino acid analysis also reveals the presence of an 18 amino acid putative signal sequence on the primary translation product which is not present on the mature S1 polypeptide.
TL;DR: To study structural variants of human serum amyloid A (SAA), an apoprotein of high-density lipoprotein, complementary DNA clones were isolated from a human liver library with the use of two synthetic oligonucleotide mixtures containing sequences that could code for residues 33-38 and 90-95 of the protein sequence.
Abstract: To study structural variants of human serum amyloid A (SAA), an apoprotein of high-density lipoprotein, complementary DNA clones were isolated from a human liver library with the use of two synthetic oligonucleotide mixtures containing sequences that could code for residues 33-38 and 90-95 of the protein sequence. The SAA-specific cDNA clone (pA1) contains the nucleotide sequence coding for the mature SAA and 10 amino acids of the 18-residue signal peptide. It also includes a 70 nucleotide long 3'-untranslated region and approximately 120 bases of the poly(A) tail. The derived amino acid sequence of pA1 is identical with the alpha form of apoSAA1. A fragment of pA1 containing the conserved (residues 33-38) region of SAA also hybridized with RNA from human acute phase liver and acute phase stimulated, but not unstimulated, mouse and rabbit liver. In contrast, a fragment corresponding to the variable region hybridized to a much greater extent with human than with rabbit or murine RNA. Human acute phase liver SAA mRNA (approximately 600 nucleotides in length) directs synthesis of preSAA (Mr 14 000) in a cell-free translating system. In a Xenopus oocyte translation system preSAA is synthesized and processed to the mature Mr 12 000 product. The complete 18 amino acid signal peptide sequence of preSAA was derived from sequencing cDNA synthesized by "primer extension" from the region of SAA mRNA corresponding to the amino terminus of the mature product. Two other SAA-specific cDNA clones (pA6 and pA10) differed from pA1 in that they lack the internal PstI restriction enzyme site spanning residues 54-56 of pA1.(ABSTRACT TRUNCATED AT 250 WORDS)
TL;DR: Phenotypes of insertion mutations isolated in the tetracycline-resistance gene support the hypothesis that it is comprised of two domains connected by a central hinge, and mutations in the beta-lactamase gene are temperature sensitive and demonstrate altered sensitivity to various beta- lactams and inhibitors.
Abstract: An efficient method for introducing two codons into a cloned gene has been applied to studying functional regions of the pBR322-encoded tetracycline-resistance gene and beta-lactamase (ampicillin-resistance) gene. Single-stranded hexameric linkers are inserted into a preexisting cohesive end restriction site to create a new (six-base recognition) restriction site. Insertion mutations are enriched by using biochemical selection or are selected by using a kanamycin-resistance cassette (biological selection). Phenotypes of insertion mutations isolated in the tetracycline-resistance gene support the hypothesis that it is comprised of two domains connected by a central hinge. Mutations in the beta-lactamase gene are temperature sensitive and demonstrate altered sensitivity to various beta-lactams and inhibitors.
TL;DR: The DNA sequences encoding 18 S ribosomal RNA in man and in the frog, Xenopus borealis and Xenopus laevis, are determined and the methyl group distribution corresponds closely to that in Xenopus, but there are several extra methyl groups in mammalian 18 S ribsomal RNA.
Abstract: We have determined the DNA sequences encoding 18 S ribosomal RNA in man and in the frog, Xenopus borealis. We have also corrected the Xenopus laevis 18 S sequence: an A residue follows G-684 in the sequence. These and other available data provide a number of representative examples of variation in primary structure and secondary modification of 18 S ribosomal RNA between different groups of vertebrates. First, Xenopus laevis and Xenopus borealis 18 S ribosomal genes differ from each other by only two base substitutions, and we have found no evidence of intraspecies heterogeneity within the 18 S ribosomal DNA of Xenopus (in contrast to the Xenopus transcribed spacers). Second, the human 18 S sequence differs from that of Xenopus by approx. 6.5%. About 4% of the differences are single base changes; the remainder comprise insertions in the human sequence and other changes affecting several nucleotides. Most of these more extensive changes are clustered in a relatively short region between nucleotides 190 and 280 in the human sequence. Third, the human 18 S sequence differs from non-primate mammalian sequences by only about 1%. Fourth, nearly all of the 47 methyl groups in mammalian 18 S ribosomal RNA can be located in the sequence. The methyl group distribution corresponds closely to that in Xenopus, but there are several extra methyl groups in mammalian 18 S ribosomal RNA. Finally, minor revisions are made to the estimated numbers of pseudouridines in human and Xenopus 18 S ribosomal RNA.
TL;DR: Biotin has been converted to 2-(biotinylamido)ethanol and condensed to phosphorylated oligonucleotides in a solid phase synthesis and the hybridization properties of such biotin-labeled oligon DNA probes were studied.
Abstract: Biotin has been converted to 2-(biotinylamido)ethanol and condensed to phosphorylated oligonucleotides in a solid phase synthesis. The 5'-biotinylated oligonucleotides were enzymatically coupled to other DNA fragments by T4 DNA ligase or T4 RNA ligase. The hybridization properties of such biotin-labeled oligonucleotide probes were studied.
TL;DR: The molecular weight difference in the polymorphic variants of the CR1 protein is based on differences in nucleotide sequences, which indicates either a series of homologous domains in a single CR1 gene or the presence of multiple CR1 genes.
Abstract: Redundant oligonucleotides were synthesized based on amino acid sequences of tryptic peptides from the purified receptor for human complement fragments C3b/C4b (CR1). These probes were used to screen a size-selected human tonsilar cDNA library. A single positive clone was identified that hybridized to three oligonucleotide probes. The cDNA insert was 1.5 kilobases in length and contained sequences homologous to those of the oligonucleotide probes as well as nucleotide sequences corresponding to another independent CR1 tryptic peptide. Blot-hybridization analysis using fragments of the cDNA insert as probes revealed two distinct species of the CR1 message of 9 and 11 kilobases in human tonsil mRNA. The two EcoRI fragments of the CR1 cDNA insert hybridized to each other, suggesting the presence of homologous sequences. When used as probes in Southern blot analysis of human DNA, each fragment identified similar but not identical patterns of multiple restriction fragments, indicating either a series of homologous domains in a single CR1 gene or the presence of multiple CR1 genes. Furthermore, an additional BamHI fragment was found to segregate with the expression of the S allotype of the CR1 protein in a family. Thus, the molecular weight difference in the polymorphic variants of the CR1 protein is based on differences in nucleotide sequences.
TL;DR: The methods and procedures described in this chapter depends upon the initial growth of the bacterial cells and the isolation of DNA or RNA from them and their importance cannot be overemphasized.
Abstract: Publisher Summary This chapter discusses the deoxyribonucleic acid (DNA) reassociation and ribonucleic acid (RNA) hybridization of bacterial nucleic acids. The elucidation of the structure and the physical properties of deoxyribonucleic acid (DNA) and ribonucleic acids (RNAs) has enabled investigators to compare directly the sequence similarities in these molecules from one organism with those from another. These comparisons, DNA reassociation or RNA hybridization experiments, are measurements of the pairing of two DNA fragments or the pairing of an RNA molecule with a fragment of DNA. The specific pairings are between the base pairs adenine (A) and thymine (T) [or uracil (U) in RNA] and between guanine (G) and cytosine (C). The final success of the methods and procedures described in this chapter depends upon the initial growth of the bacterial cells and the isolation of DNA or RNA from them. Although techniques for following the growth of bacterial cultures are beyond the scope of this chapter, their importance cannot be overemphasized. Many organisms are fastidious and grow very slowly or to a low turbidity, others tend to die very rapidly after reaching stationary phase, while others may enter a sporulation phase.
TL;DR: Transfection experiments showed that only those recombinant clones with the mutation in position 61 were biologically active, and predicted a glutamine-to-lysine substitution in amino acid 61, a change confirmed by conventional sequencing of the first and second exons of N-ras from cell line Mel Swift.
Abstract: DNA isolated from cell line Mel Swift, a human melanoma cell line, transforms NIH3T3 cells. Southern blot analysis of DNA from secondary foci revealed conserved 8.8- and 7.8-kilobase EcoRI fragments which hybridized with a human repetitive sequence clone, blur 8. The activated transforming gene was identified as N-ras, and the 8.8-kilobase EcoRI fragment from a secondary transformant was cloned. Synthetic 17-mer oligonucleotides which spanned either the normal codon 61 (CAA) or a mutant codon 61 (AAA) were used for hybridization. Cloned N-ras from melanoma cell line Mel Swift hybridized to the mutant (AAA) oligonucleotide. From this we predicted a glutamine-to-lysine substitution in amino acid 61, a change confirmed by conventional sequencing of the first and second exons of N-ras from cell line Mel Swift. Transfection experiments showed that only those recombinant clones with the mutation in position 61 were biologically active.