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I G Wool

Publications and source records attributed to I G Wool.

At least 109 records · Page 6Linked to original sources
PubMed1984

The nucleotide sequence of a rat 18 S ribosomal ribonucleic acid gene and a proposal for the secondary structure of 18 S ribosomal ribonucleic acid.

The nucleotide sequence of a rat 18 S rRNA gene was determined. The 18 S rRNA encoded in the gene contains 1874 nucleotides, and the molecular weight estimated from the sequence is 6.09 X 10(5). The sequences of rat and Xenopus laevis 18 S rRNAs are very similar; the only differences of consequence are the insertions between nucleotides 197 and 206 and between 258 and 279 of the rat nucleic acid of sequences rich in guanine and cytosine. A proposal is presented for the secondary structure of rat 18 S rRNA based on a comparison with the sequences of 17 other small ribosomal subunit RNAs. While the primary sequences of rat and eubacterial RNAs are different, the deduced secondary structures are remarkably similar.

Animals↗
PubMed1984

The primary structure of rat liver ribosomal protein L39.

The covalent structure of the rat liver 60 S ribosomal subunit protein L39 was determined. Fourteen tryptic peptides were purified, and the sequence of each was established by a micromanual procedure; they accounted for all 50 residues of L39. The sequence of the NH2-terminal 32 residues of L39, obtained by automated Edman degradation of the intact protein, provided the alignment of the first seven tryptic peptides. Two peptides, CNI (28 residues) and CNII (22 residues), were produced by cleavage of protein L39 with cyanogen bromide and the sequence of CNII was determined by automated Edman degradation. This sequence established the order of tryptic peptides T8 through T14. The carboxyl-terminal amino acids were identified after carboxypeptidase A treatment. Protein L39 contains 50 amino acids and has a molecular weight of 7308. There are indications that a portion of rat L39 is related to a fragment of Escherichia coli ribosomal protein S1.

Animals↗
PubMed1984

Nuclease protection analysis of ribonucleoprotein complexes: use of the cytotoxic ribonuclease alpha-sarcin to determine the binding sites for Escherichia coli ribosomal proteins L5, L18, and L25 on 5S rRNA.

A rapid and convenient method has been devised to determine the binding sites for proteins on RNA. The procedure is an adaptation of one used to map DNA-protein complexes by protection against nuclease digestion. The method uses the cytotoxic ribonuclease alpha-sarcin, which hydrolyzes purines in both single- and double-stranded regions of RNA. It has been authenticated by confirming the binding sites for the Escherichia coli ribosomal proteins L18 and L25 on 5S rRNA and its value has been established by identifying the attachment site for protein L5. The procedure should be useful for the analysis of other ribonucleoprotein complexes.

Binding Sites↗
PubMed1983

The structure of rat 28S ribosomal ribonucleic acid inferred from the sequence of nucleotides in a gene.

The nucleotide sequence of a rat 28S rRNA gene was determined. The 28S rRNA encoded in the gene contains 4718 nucleotides and the molecular weight estimated from the sequence is 1.53 x 10(6). The guanine and cytosine content is 67%. The sequence of rat 28S rRNA diverges appreciably from that of Saccharomyces carlsbergensis 26S rRNA (about 50% identity), but more closely approximates that of Xenopus laevis 28S rRNA (about 75% identity). Rat 28S rRNA is larger than the analogous nucleic acids from yeast (3393 nucleotides) and X, laevis (4110 nucleotides) ribosomes. The additional bases are inserted in specific regions and tend to be rich in guanine and cytosine. 5.8S rRNA can interact with 28S rRNA by extensive hydrogen bonding at two sites near the 5' end of the latter.

Animals↗
PubMed1983

The sequence of the nucleotides at the alpha-sarcin cleavage site in rat 28 S ribosomal ribonucleic acid.

The sequence of the 521 nucleotides at the 3' end of a rat 28 S rRNA gene was determined. The region encompasses the site of cleavage of 28 S rRNA by the cytotoxin alpha-sarcin. The toxin hydrolyzes a phosphodiester bond on the 3' side of a guanine residue 393 nucleotides from the 3' end. The alpha-sarcin domain is composed of a purine-rich sequence of 14 highly conserved nucleotides.

Animals↗
PubMed1983

The primary structure of rat liver ribosomal protein L37. Homology with yeast and bacterial ribosomal proteins.

The covalent structure of the rat liver 60 S ribosomal subunit protein L37 was determined. Twenty-four tryptic peptides were purified and the sequence of each was established; they accounted for all 111 residues of L37. The sequence of the first 30 residues of L37, obtained previously by automated Edman degradation of the intact protein, provided the alignment of the first 9 tryptic peptides. Three peptides (CN1, CN2, and CN3) were produced by cleavage of protein L37 with cyanogen bromide. The sequence of CN1 (65 residues) was established from the sequence of secondary peptides resulting from cleavage with trypsin and chymotrypsin. The sequence of CN1 in turn served to order tryptic peptides 1 through 14. The sequence of CN2 (15 residues) was determined entirely by a micromanual procedure and allowed the alignment of tryptic peptides 14 through 18. The sequence of the NH2-terminal 28 amino acids of CN3 (31 residues) was determined; in addition the complete sequences of the secondary tryptic and chymotryptic peptides were done. The sequence of CN3 provided the order of tryptic peptides 18 through 24. Thus the sequence of the three cyanogen bromide peptides also accounted for the 111 residues of protein L37. The carboxyl-terminal amino acids were identified after carboxypeptidase A treatment. There is a disulfide bridge between half-cystinyl residues at positions 40 and 69. Rat liver ribosomal protein L37 is homologous with yeast YP55 and with Escherichia coli L34. Moreover, there is a segment of 17 residues in rat L37 that occurs, albeit with modifications, in yeast YP55 and in E. coli S4, L20, and L34.

Amino Acid Sequence↗
PubMed1983

The primary structure of the cytotoxin alpha-sarcin.

The primary structure of the cytotoxin alpha-sarcin was determined. Eighteen of the 19 tryptic peptides were purified; the other peptide has arginine only. The complete sequence of 17 of the peptides was determined; the sequence of the remaining peptide was determined in part. The sequence of the 39 NH2-terminal residues was obtained by automated Edman degradation. The carboxyl-terminal amino acids were identified after carboxypeptidase treatment. The assignment of the amino acids in the tryptic peptides was confirmed and their alignment established from the sequence of the secondary tryptic peptides obtained after cleavage of citraconylated alpha-sarcin, from the sequence of a 2-(2-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine peptide, from the sequence of a chymotryptic peptide, and from the sequence of a peptide obtained with Staphylococcus aureus V8 protease. alpha-Sarcin contains 150 amino acid residues; the molecular weight is 16,987. There are disulfide bridges between cysteine residues at positions 6 and 148 and between residues 76 and 132.

Amino Acid Sequence↗
PubMed1983

The ribonuclease activity of the cytotoxin alpha-sarcin. The characteristics of the enzymatic activity of alpha-sarcin with ribosomes and ribonucleic acids as substrates.

The ribonuclease activity of the cytotoxic protein alpha-sarcin has been characterized. When rat liver ribosomes or 60 S ribosomal subunits were the substrates, alpha-sarcin cleaved a single oligonucleotide of about 488 residues, the alpha-fragment, from the 3' end of 28 S rRNA. In contrast, 40 S ribosomal subunits were not affected by alpha-sarcin. The alpha-fragment was cleaved from 28 S rRNA in 80 S ribosomes when the concentration of alpha-sarcin was 3 x 10(-8) M and the toxin retained its specificity even when the concentration was 3 x 10(-5) M. The turnover number (kcat) for the reaction of alpha-sarcin with ribosomes was 55 min-1, establishing that the toxin acts catalytically. When total rRNA or 28 S rRNA was the substrate, alpha-sarcin caused extensive progressive digestion of the nucleic acids; however, no formation of the alpha-fragment occurred. The extent of the digestion of 28 S rRNA was related to the concentration of alpha-sarcin, but the amount of the toxin required was somewhat greater than that needed with ribosomes. Digestion of homopolynucleotides with alpha-sarcin indicated that the protein is specific for purines. When [32P]5 S rRNA was the substrate, alpha-sarcin cleaved on the 3' side of purines in both single- and double-stranded regions of the molecule. The results suggest that the unusual specificity of alpha-sarcin, in that it cleaves only one of more than 7000 phosphodiester bonds in the ribosome, is a property both of the cytotoxin and of the ribosome.

Animals↗
PubMed1983

Location of 5.8 S rRNA contact sites in 28 S rRNA and the effect of alpha-sarcin on the association of 5.8 S rRNA with 28 S rRNA.

We have constructed phage M13mp7 clones each containing the coding strand from one of three restriction fragments which collectively span the mouse 28 S rRNA gene with the exception of the 3'-terminal approximately 500 base pairs. When hybridized to 28 S rRNA, only the fragment containing the 5'-terminal 1400 nucleotides of the gene inhibited the annealing of 5.8 S rRNA to the 28 S rRNA. The same results were obtained when either the 5'- or 3'-terminal fragment of 5.8 S rRNA was used in lieu of intact 5.8 S rRNA, each of which had been shown to contain an independent 28 S rRNA contact site. However, alpha-sarcin, a cytotoxin that inhibits protein synthesis by hydrolyzing a phosphodiester bond near the 3' end of 28 S rRNA, produces a 3'-terminal 488-nucleotide fragment which exhibits a marginal capacity to anneal to 5.8 S rRNA. These results indicate that 5.8 S rRNA interacts predominantly with a structural domain near the 5' end of 28 S rRNA. This conclusion is consistent with base-pairing interactions between 5.8 S rRNA and 28 S rRNA based on the proposed secondary structures for Escherichia coli 23 S and yeast 26 S rRNAs. However, alpha-sarcin treatment of ribosomes affects the stability of the binding of 5.8 S rRNA to the 28 S rRNA, even though the toxin hydrolyzes a phosphodiester bond several thousand nucleotides from the proposed contact regions. Finally, mouse 5.8 S rRNA was shown to lack two internal nucleotides reported to be present in rat 5.8 S rRNA.

Animals↗
PubMed1982

Separate binding sites on rat liver ribosomal protein L6 for 5 S and 5.8 S ribosomal ribonucleic acids and for transfer ribonucleic acids.

The rat liver ribosomal protein L6 binds to 5 S and 5.8 S rRNAs, and to initiator and elongator tRNAs. Experiments were carried out to determine if the protein has separate domains for binding each of these nucleic acids. For that purpose, nucleic acid.L6 complexes were immobilized on Sepharose and their capacity to retain 32P-labeled nucleic acids was assessed. A 5 S rRNA.L6 affinity complex binds 5.8 S [32P]rRNA indicating that L6 has separate binding sites for 5 S and 5.8 S rRNAs. A 5 S rRNA.L6 affinity complex also binds [32P]tRNAPhe, and unlabeled 5.8 S rRNA does not compete with the radioactive tRNA for binding to L6, suggesting that the ribosomal protein has a third, distinct, nucleic acid-binding domain. To determine if L6 has separate sites for the binding of elongator and initiator tRNAs, tRNAPhe. L6 and tRNAfMet.L6 affinity columns were constructed. The tRNAPhe.L6 affinity complex binds [32P]tRNAfMet, and the tRNAfMet.L6 complex binds [32P]tRNAPhe, suggesting there are distinct sites on L6 for the interaction with initiator and elongator tRNAs; however, competition experiments imply that, while there are two sites for binding tRNAs to L6, the sites do not discriminate between initiator and elongator tRNAs.

Animals↗
PubMed1982

The site of action of alpha-sarcin on eukaryotic ribosomes. The sequence at the alpha-sarcin cleavage site in 28 S ribosomal ribonucleic acid.

alpha-Sarcin is a cytotoxic protein that inhibits eukaryotic elongation factor 1 catalyzed binding of aminoacyl-tRNA to eukaryotic ribosomes as a result of cleaving a fragment from the large RNA in the 60 S ribosomal subunit. Rat liver polysomes are sensitive to alpha-sarcin in vitro only if the treatment is carried out in the presence of EDTA. The oligonucleotide produced by alpha-sarcin action on rat liver ribosomes is derived from the 3' end of 28 S rRNA and has a molecular weight of approximately 161,000 (about 488 bases). The sequence at the site of cleavage (the 5' end of the oligonucleotide produced by alpha-sarcin action on rat liver ribosomes) was AGGAAC for both rat liver 28 S rRNA and yeast 25 S rRNA. Based on the size of the alpha-fragment derived from Escherichia coli ribosomes (about 243 nucleotides), it is likely that a similar sequence, AGGACC, occurs at the site of cleavage of prokaryotic 23 S rRNA. It is also likely that the sequence on the 5' side of the alpha-sarcin cleavage site is UAGUACGAG. The sequence UAGUACGAGAGGAAC must be important because it is conserved and because hydrolysis of a single phosphodiester bond there inactivates the ribosome. We suggest that this region of the large nucleic acid in the 60 S subparticle is involved in binding aminoacyl-tRNA to the ribosome, first, because it is the eukaryotic elongation factor 1 catalyzed reaction that is inhibited by alpha-sarcin, and second, because peptidyl-tRNA seems to protect polysomes against the toxin. The function of EDTA appears to be to remove peptidyl-tRNA since puromycin also sensitizes polysomes to alpha-sarcin.

Animals↗
PubMed1982

The primary structure of the acidic phosphoprotein P2 from rat liver 60 S ribosomal subunits. Comparison with ribosomal 'A' proteins from other species.

The primary structure of rat liver ribosomal protein P2 was deduced from the sequence of the peptides. Ten peptides were obtained by cleavage of P2 with trypsin. The peptides, which accounted for the 111 residues of P2, were isolated by high voltage electrophoresis and chromatography on cellulose thin layer sheets, and the partial or complete sequence was determined by micromanual or solid-phase procedures using 4-N,N-dimethylaminoazobenzene 4'-isothiocyanate and phenylisothiocyanate. In a similar manner, the sequence of 14 peptic peptides was determined. The sequence of the NH2-terminal 30 residues of P2 was obtained by automatic Edman degradation in a sequenator. The ordering of the tryptic peptides was aided by determination of the partial or complete sequence of fragments generated with chymotrypsin, or Armillaria mellea protease, or by secondary cleavage of peptic peptides with trypsin. The carboxyl-terminal sequence was obtained from a cyanogen bromide fragment and from hydrolysis with carboxypeptidase. The sequence of protein P3 was also determined. P3 differs from P2 only in that it lacks the carboxyl-terminal 8 residues, and hence, it is likely to be a proteolytic product of P2. Rat liver ribosomal protein P2 is homologous with yeast YP A1, with Artemia salina eL12, and with Halobacterium cutirubrum L20. It is likely that rat liver P2 is also homologous with the prokaryotic ribosomal "A" proteins, Escherichia coli L7/L12, Micrococcus lysodeikticus MA1, and Bacillus subtilis L9, but that during evolution, a transposition of a portion of the molecule occurred.

Amino Acid Sequence↗
PubMed1982

The binding of transfer ribonucleic acids to 5 S and 5.8 S eukaryotic ribosomal ribonucleic acid-protein complexes.

Rat liver 5 S and 5.8 S rRNAs were oxidized with periodate and the 3' termini were coupled to Sepharose 4B through an adipic acid dihydrazide spacer. Ribosomal proteins were passed through the nucleic acid affinity columns to form ribonucleoprotein complexes containing the nucleic acid and the proteins that bind to it (5 S . L6, L7, L19; and 5.8 S . L6, L19, S9, S13). Pure isoaccepting species of yeast initiator-tRNA (tRNAfMet) and elongator-tRNAs (tRNAmMet, and tRNAPhe) were chromatographed on the ribosomal ribonucleoprotein affinity columns. The three rRNAs were bound to the 5 S and 5.8 S ribosomal ribonucleoprotein complexes. The elongation and initiation ternary complexes, EF-1 alpha . GTP . Phe-tRNAPhe, and eIF-2 . GTP . Met-tRNAfMet, also were bound to both ribosomal ribonucleoprotein affinity columns, whereas the binary complex EF-1 alpha . GTP and puromycin were not.

Animals↗
PubMed1981

Characterization of the binding of rat liver ribosomal proteins L6, L8, L19, S9, and S13 to 5.8 S ribosomal ribonucleic acid.

The interaction of rat liver ribosomal proteins L6, L8, L19, S9, and S13 with 5.8 rRNA was characterized by nitrocellulose membrane filtration. Binding approached saturation with the five proteins; the apparent association constants (K'a), measured at 4 degrees C and 22 degrees C, ranged from 0.2 to 18 X 10(5) M-1. The molar ratio of ribosomal protein and rRNA in the complexes at saturation approximated 1, indicating there is one binding site for each of the five proteins on the nucleic acid. A number of ribosomal proteins, including some previously suspected from affinity chromatography of associating weakly, did not form a complex with 5.8 S rRNA.

Animals↗
PubMed1980

Characterization of the binding of rat liver ribosomal proteins L6, L7, and L19 to 5 S ribosomal ribonucleic acid.

The binding of rat liver ribosomal proteins L6, L7, and L19 to 5 S rRNA was characterized by nitrocellulose membrane filtration. Binding could be saturated with the three proteins; the apparent association constants (Ka'), measured at 4 degrees C and 22 degrees C, ranged from 1.3 to 6.8 x 10(5) M-1. The molar ratio of ribosomal protein and rRNA in the complex at saturation approximated 1, indicating there is one binding site for each of the three proteins on the nucleic acid. A large number of rat liver ribosomal proteins, including some previously suspected of associating weakly, did not form a complex with 5 S rRNA.

Animals↗
PubMed1980

The identification by affinity chromatography of the rat liver ribosomal proteins that bind to elongator and initiator transfer ribonucleic acids.

Mixed yeast elongator-tRNAs (bulk tRNA lacking fRNAm,fMet), pure isoaccepting species of elongator-tRNAs (tRNAmMet and tRNAPhe), and purified initiator-tRNA (tRNAfMet) were each oxidized with periodate and the 3' terminus was coupled to Sepharose 4B through an adipic acid dihydrazide spacer. The rat liver ribosomal proteins that associated with the tRNAs were isolated by affinity chromatography and identified by electrophoresis in polyacrylamide gels. The rat liver ribosomal proteins that were bound to the elongator-tRNA preparations were L6, L35a, and S15; small amounts of a number of other proteins also associated with the nucleic acid. When initiator-tRNA (tRNAfMet) was immobilized on Sepharose, only L6 and L35a were bound; no 40 S subunit proteins associated with initiator-tRNA. No Escherichia coli proteins formed a complex with either eukaryotic initiator- or elongator-tRNAs.

Animals↗
PubMed1980

The effect of antibodies against Escherichia coli small ribosomal subunit proteins on protein synthesis by rat liver ribosomes.

Monovalent Fab fragments from immunoglobulins, directed against Escherichia coli small ribosomal subunit proteins, were tested for their effect on poly(U)-directed synthesis of polyphenylalanine by rat liver ribosomes. Of the 18 Fabs tested, 3, anti-S10, anti-S12, and anti-S14, inhibited polyphenylalanine synthesis; antibodies against 3 of the 21 E. coli ribosomal proteins (S1, S16, and S17) were not tested. The others were either without any appreciable effect or, as in the case of anti-S11 and anti-S18, inhibited far less. Antibodies against S12 and S14 (but not against S10) inhibited elongation factor 1-dependent binding of [14C]Phe-tRNA to rat liver 80 S ribosomes. The presumption is that the prokaryotic (E. coli) ribosomal proteins, S10, S12, and S14, possess antigenic determinants also present in eukaryotic (rat liver) ribosomal proteins; the rat liver proteins have not yet been identified.

Animals↗