Specificity of oligo (dT)-cellulose chromatography in the isolation of polyadenylated RNA

Bantle, John A.; Maxwell, Ian H.; Hahn, William E.

doi:10.1016/0003-2697(76)90549-2

Cited by 360 publications

(127 citation statements)

References 16 publications

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“…Poly(A)-rich RNA was purified by affinity chromatography using oligo(dT)-cellulose [13]. Polysomes and RNAs were translated in vitro using wheat-germ cell-free extracts supplemented with [3H]leucine and [3H]proline [14].…”

Section: Plant Materialsmentioning

confidence: 99%

Nucleotide sequence of barley chymotrypsin inhibitor‐2 (CI‐2) and its expression in normal and high‐lysine barley

Williamson

Forde

Buxton

et al. 1987

European Journal of Biochemistry

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cDNA clones for chymotrypsin inhibitor-2 (CI-2) have been isolated from an endosperm-specific library of barley using a synthetic oligonucleotide probe. The nucleotide sequences of several of the cDNAs predict an open reading frame (beginning with an ATG codon) which encodes a protein of 84 residues (Mr 9380). In the longest clone another ATG codon is present, a further 69 nucleotides upstream. The nucleotide sequence between these two ATG codons predicts an amino acid sequence with the characteristics of a signal peptide, as found in other cloned plant protease inhibitors. However, it contains an in-frame TAA stop codon, which is also present in all of the shorter cDNAs which extend into this region. From in vitro translation experiments, using mRNAs synthesized from cDNAs, we conclude that, in vitro, translation of all or the vast majority of CI-2 mRNAs begins at the second ATG codon, 31 nucleotides downstream from the ochre stop codon.Southern blotting of genomic DNA shows that CI-2 is encoded by a small multigene family, while sequence analysis of the cDNAs shows that at least two sub-families of mRNAs, which are more than 90% homologous, are present in the endosperm. Northern blotting analysis shows that related but different sequences are present in leaf and shoot RNA populations.Further Northern blot hybridizations using RNA from the normal line, Sundance, and the high-lysine barley mutant, Hiproly, show that endosperms of the latter contain greatly increased levels of CI-2 mRNA. This correlates with the increased amount of CI-2 protein deposited in Hiproly, and demonstrates that the differential expression of (21-2 in the two genotypes is controlled at the level of transcription and/or stability of the mRNA. In contrast, the abundance of CI-2 mRNAs in leaves and shoots is not affected.Barley endosperm contains two chymotrypsin inhibitors called CI-1 and CI-2 which belong to a family of proteins including the potato inhibitor I and the leech inhibitor elgin [l]. CI-2 normally accounts for about 0.25% of the total salt-soluble proteins of the barley grain (calculated from [2]). However, the spontaneous high-lysine barley mutant Hiproly [3] has a 20-fold increased content of CI-2 [4, 51. Because of its high lysine content (11.5 g YO), CI-2 accounts for 37% of the difference in lysine content between Hiproly and normal barley grains and so contributes significantly to the improved nutritional quality of Hiproly. The protein has therefore received considerable attention. It has been purified from Hiproly endosperms and characterized in detail [5 -71.The increased amount of CI-2 in Hiproly is controlled by a single recessive gene called lys on chromosome 7 [3, 81, whereas the structural genes for CI-2 are located at the Zca-2 locus on chromosome 5 [9]. It is not known how the product of the lys gene affects the regulation of expression of the Zca-2 locus, though a recent study by Rasmussen [lo] suggests that the increased amount of CI-2 in Hiproly may stem from an earlier synthesis of CI-2 during endosperm de...

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Section: Plant Materialsmentioning

confidence: 99%

Nucleotide sequence of barley chymotrypsin inhibitor‐2 (CI‐2) and its expression in normal and high‐lysine barley

Williamson

Forde

Buxton

et al. 1987

European Journal of Biochemistry

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“…(13), and the poly(A) RNA fraction was recovered from total polysomal RNA by three passes through an oligo(dT)-cellulose column (type 7; PL Biochemicals, Milwaukee, WI) by the procedure of Bantle et al (1).…”

mentioning

confidence: 99%

Synthesis of Oat Globulin Precursors

Brinegar

Peterson²

1982

Plant Physiol.

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The oat (Avena sativa L.) seed globulin was found to be synthesized in vitro as 60,000 to 64,000 dalton precursors. In vivo protein labeling yielded polypeptides of 58,000 to 62,000 daltons, suggesting cleavage of signal sequences from the precursors. Further cleavage is apparently required to separate the a and ,t polypeptide sequences which are known to form disulfide-linked 53,000 to 58,000 dalton species in the (af)6 holoprotein.The data are discussed with respect to analogous synthesis and processing of some legume 11S storage proteins.The major storage protein of oat seeds is a globulin which comprises as much as 75% of the total seed protein, whereas only 10% is accounted for by the alcohol-soluble prolamin avenin (18). Such a distribution is unusual among cereals, in which the prolamin fraction generally predominates (10). In a recent study, we showed the globulin consisted of heterogeneous sets of a and f8 polypeptides of 32,500 to 37,500 and 22,000 to 24,000 D, and isoelectric points of 5.9 to 7.2 and 8.7 to 9.2, respectively (3). Electrophoretic analysis demonstrated that the a and ,B polypeptides were disulfide-linked in vivo, forming a,8 species of mol wt 53,000 to 58,000 daltons. Based on the holoprotein stoichiometry of six each of the a and ,B polypeptides proposed by Peterson (17), we suggested a native globulin structure of (a,8)6 with an average

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“…Polyadenylated RNA was prepared by affinity chromatography on oligo(dT)-cellulose (Bantle et al 1976). …”

Section: Methodsmentioning

confidence: 99%

The Isolation and Characterisation of the Ovine Growth Hormone Gene

Byrne¹,

Wilson²,

Ward³

1987

Aust. Jnl. Of Bio. Sci.

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The ovine growth hormone gene has been isolated and sequenced, together with about 1 kbp of DNA flanking each end of the gene. The structure of the gene is similar to that found for other growth hormone genes, particularly the bovine gene, and has a primary transcript of 1792 bp, with five exons, and with intron sizes of 264 bp, 231 bp, 227 bp and 273 bp.The gene is flanked by artiodactyl-specific middle-repetitive DNA, consisting mainly of elements belonging to the 'C-A3' family of repeated DNA. A previously unreported sequence has been found, which we have named an 'E' element.

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Specificity of oligo (dT)-cellulose chromatography in the isolation of polyadenylated RNA

Cited by 360 publications

References 16 publications

Nucleotide sequence of barley chymotrypsin inhibitor‐2 (CI‐2) and its expression in normal and high‐lysine barley

Nucleotide sequence of barley chymotrypsin inhibitor‐2 (CI‐2) and its expression in normal and high‐lysine barley

Synthesis of Oat Globulin Precursors

The Isolation and Characterisation of the Ovine Growth Hormone Gene

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