Rapid synthesis of oligodeoxyribonucleotides VI. Effident, mechanised synthesis of heptadecadeoxyribonucleotides by an improved solid phase phosphotriester route

Duckworth, Mary Lynn; Gait, Michael J.; Goelet, Philip; Hong, Guo; Singh, Mandeep; Titmas, Richard C.

doi:10.1093/nar/9.7.1691

Cited by 190 publications

(61 citation statements)

References 13 publications

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“…M13ED1 is a M13mp8 (Messing and Vieira, 1982) vector carrying a 160-base fibronectin insert, corresponding to a region within the ED (positions 2004 -2164 in Figure 2 of Kornblihtt et al, 1984b (Messing et al, 1981). The orientation of the ED insert is such that extension of a 'universal' sequencing primer (Duckworth et al, 1981) with Klenow polymerase using M13ED I DNA as template results in a single-stranded probe com-nplementary to ED mRNA sequences.…”

Section: Isolation Of X Clonesmentioning

confidence: 99%

Expression of a human alpha-globin/fibronectin gene hybrid generates two mRNAs by alternative splicing.

Vibe-Pedersen¹,

Kornblihtt²,

Baralle³

1984

The EMBO Journal

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We have isolated genomic clones for human fibronectin (FN (Kornblihtt et al., 1983(Kornblihtt et al., ,1984a(Kornblihtt et al., ,1984b. The inserts of these clones were used as probes to screen a human gene library in bacteriophage X. Six positive plaques were identified. The plaques represent at least two independent clones (see Materials and methods) named XFN4 (15.8 kb) and XFN5 (15.3 kb). Both contain the extra domain (ED) (Figure lA).Restriction enzyme mapping by Southern blotting showed that the ED and the adjacent regions of the cDNA were contained in a 4.3-kb EcoRI fragment of XFN5. This fragment was sub-cloned and the resulting recombinant plasmid (pFN5RI 4.3) was mapped in detail ( Figure IC). The nucleotide sequence of its exons and of the exon/intron boundaries was determined as outlined in Figure IC. The extra domain ED corresponds to precisely one exon (270 bp), which encodes a full type III homology, whereas the exon just 5' to the ED (exon -1) and just 3' to it (exon + 1) are shorter (114 and 188 bp, respectively) and encode only part of a type III unit. The sequences of the exon/intron boundaries are shown in Figure 2. The sequence of the exons (not shown) and that of the corresponding area in the cDNA clones (Kornblihtt et al., 1984a) are identical, except for one base exchange. This variation does not change any amino acids, but creates a restriction enzyme polymorphism since an XhoI site (CTCGAG) is present in the middle of the ED in the cDNA clones, but absent (CTCTAG) in the ED exon of the isolated genomic clones.Southern blot analysis of restriction enzyme digests (results not shown) of both genomic clones using several cDNA probes allowed us to draw the map of part of the human FN gene shown in Figure lB. XFN4 and XFN5 overlap by 2.7 kb and together span a central 28-kb portion of the FN gene. Although the size of the human gene has not been determined, it is likely to be similar to that of the chicken FN gene (-50 kb) (Hirano et al., 1983). The approximate position of the exons -3, -2 and + 2, + 3 on the gene map ( Figure 1B (pSVED-2511

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Section: Isolation Of X Clonesmentioning

confidence: 99%

Expression of a human alpha-globin/fibronectin gene hybrid generates two mRNAs by alternative splicing.

Vibe-Pedersen¹,

Kornblihtt²,

Baralle³

1984

The EMBO Journal

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“…A mixture of 17-nucleotide-long oligonucleotides encoding a portion of the rat liver cathepsin B protein sequence was synthesized by using a solid-phase phosphotriester method (14,15). For amino acids specified by more than one codon, all possible nucleotides were inserted at the ambiguous positions.…”

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confidence: 99%

Isolation of a cDNA clone for the human lysosomal proteinase cathepsin B.

Fong¹,

Calhoun²,

Hsieh³

et al. 1986

Proc. Natl. Acad. Sci. U.S.A.

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The cysteine proteinase cathepsin B is one member of the lysosomal acid hydrolases. Based on the peptide sequence of rat liver cathepsin B, an oligonudeotide 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 done encodes human cathepsin B. The clone, designated pCB-1, has sequences for 81% of the coding region (for amino acid residues 50-252) together with w880 nucleotides of the 3' untranslated region of the mRNA. The DNA sequence also shows that the predicted carboxyl terminus of the coding sequence is longer than the mature protein by 6 amino acid residues. Southern blot analysis of restriction enzyme digests of human placental DNA revealed a simple pattern of hybridizing fragments using the cathepsin B coding sequence as probe. The result suggests that there is a single copy of cathepsin B gene per haploid genome.Proteinases are present in all forms of living organisms. The events of general and limited proteolyses have been attributed to actions of these enzymes. In general proteolysis, proteinases digest nutrient proteins and participate in the turnover of cellular proteins, whereas in limited proteolysis, proteinases modify substrate proteins and alter the properties and physiological functions of these proteins. Thus, limited proteolysis can play regulatory roles during cell growth and differentiation (1,2).Based on the amino acid residues at their active sites, proteinases are classified into serine, cysteine, aspartate, and metalloproteinases. Members of the cysteine proteinase family have wide phylogenetic distribution, and examples include papain from the papaya plant and cathepsin B from mammalian tissues. Cathepsin B is a lysosomal enzyme that is functional during intracellular protein catabolism and may also be involved in the proteolytic processing of protein precursors such as proinsulin (3)(4)(5). Cathepsin B has been implicated in several disease states including muscular dystrophy (6), rheumatoid arthritis (7), and tumor metastasis (8-11). We and others have demonstrated that cathepsin B-like proteinases may also be involved during differentiation of the cellular slime mold Dictyostelium discoideum (12, 13).To further investigate the role of cathepsin B in cellular functions, we have selected the molecular genetic approach. Here we report our results on the isolation and characterization of a cDNA clone for human cathepsin B.MATERIALS AND METHODS Synthesis of Oligodeoxyribonucleotides. A mixture of 17-nucleotide-long oligonucleotides encoding a portion of the rat liver cathepsin B protein sequence was synthesized by using a solid-phase phosphotriester method (14, 15). For amino acids specified by more than one codon, all possible nucleotides were inserted at the ambiguous positions. The synthesis was performed with a Vega Model 280 Automated Polynucleotide Synthesizer by a...

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“…Single-stranded templates of M13 were isolated (Birnboim & Doly, 1979) and sequenced by the dideoxy chain termination method (Sanger et al, 1977) using the universal primer 5' d(GTAAAACGACGGCCAGT) 3' (BRESA) (Duckworth et al, 1981). The reaction was carried out at 50 °C (Gomer et al, 1985) to overcome problems with long homopolymeric sequences.…”

Section: Methodsmentioning

confidence: 99%

Nucleotide Sequence of the Capsid and Nuclear Inclusion Protein Genes from the Johnson Grass Strain of Sugarcane Mosaic Virus RNA

Gough

Azad

Hanna

et al. 1987

Journal of General Virology

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Rapid synthesis of oligodeoxyribonucleotides VI. Effident, mechanised synthesis of heptadecadeoxyribonucleotides by an improved solid phase phosphotriester route

Cited by 190 publications

References 13 publications

Expression of a human alpha-globin/fibronectin gene hybrid generates two mRNAs by alternative splicing.

Expression of a human alpha-globin/fibronectin gene hybrid generates two mRNAs by alternative splicing.

Isolation of a cDNA clone for the human lysosomal proteinase cathepsin B.

Nucleotide Sequence of the Capsid and Nuclear Inclusion Protein Genes from the Johnson Grass Strain of Sugarcane Mosaic Virus RNA

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