The chain termini of polynucleotides formed by limited enzymic fragmentation of wheat embryo ribosomal RNA. Part 1. Studies of snake venom phosphodiesterase

1968 ◽  
Vol 46 (1) ◽  
pp. 81-92 ◽  
Author(s):  
B. D. McLennan ◽  
B. G. Lane
Keyword(s):  
Snake Venom ◽  
Ribosomal Rna ◽  
Sedimentation Rate ◽  
Sharp Decrease ◽  
Degree Of Polymerization ◽  
Wheat Embryo ◽  
Snake Venom Phosphodiesterase ◽  
Endonucleolytic Cleavage ◽  
Limited Degree ◽  
Hydrolysis Of

Snake venom phosphodiesterase induces about fifteen exonucleolytic cleavages for each endonucleolytic cleavage during the first hour of hydrolysis of wheat embryo ribosomal RNA, under the conditions of hydrolysis used in this present investigation. The polynucleotide chains in the ribosomal RNA preparation have an average degree of polymerization in the neighborhood of 1300 nucleotide residues, and there is a mean of between 5 and 10 endonucleolytic breaks per chain during this first hour of phosphodiesterase-induced hydrolysis. The cleavages occur widely throughout most of the polynucleotide chains in the ribosomal RNA preparation, as judged by the sharp decrease in mean sedimentation rate which accompanies a limited degree (about 10%) of exonucleolysis of the RNA. Studies of phosphodiesterase-induced endonucleolysis of wheat embryo soluble RNA are reported, but because of the much lower initial degree of polymerization (about 80 nucleotide residues per polynucleotide chain), the results of endonucleolysis are less pronounced in terms of the proportional increment in chain termini and the proportional decrease of mean sedimentation rate. The endonucleolysis of RNA is discussed in terms of the minor nucleotide components in both ribosomal and soluble RNA, and particular reference is made to pseudouridylate which has been found in relatively high proportion among the chain termini after limited hydrolysis with venom phosphodiesterase.Purified venom phosphodiesterase preparations, devoid of ribonuclease or 5′-nucleotidase contamination, were found to convert nucleoside 2′(3′),5′-diphosphates to 5′-nucleotides under conditions which had virtually no effect on nucleoside 3′-phosphates or nucleoside 5′-phosphates. The possibility that this reaction may be catalyzed by the venom phosphodiesterase itself is discussed.

Analysis of O2′-Methylnucleoside 5′-Phosphates in Snake Venom Hydrolysates of RNA: Identification of O2′-Methyl-5-Carboxymethyluridine as a Constituent of Yeast Transfer RNA

1975 ◽  
Vol 53 (7) ◽  
pp. 735-746 ◽  
Author(s):  
M. W. Gray
Keyword(s):  
Snake Venom ◽  
Deae Cellulose ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Assay Method ◽  
Blocking Group ◽  
Wheat Embryo ◽  
Yeast Trna ◽  
Snake Venom Phosphodiesterase ◽  
Hydrolysis Of

Snake venom phosphodiesterase liberates the O2′-methylnucleoside (Nm) constituents of RNA as the corresponding 5′-nucleotides (pNm), which, in contrast to normal 5′-nucleotides (pN), are resistant to dephosphorylation by venom 5′-nucleotidase. This property provides the basis of a convenient and highly reproducible quantitative assay for Nm residues in RNA. The assay method involves: (1) hydrolysis of RNA with whole or partially-purified snake venom; (2) isolation of the pNm derivatives, as a group, by anion-exchange chromatography on DEAE-cellulose; (3) resolution of the individual pNm compounds by two-dimensional paper chromatography; (4) identification and quantitative measurement of pNm derivatives by ultraviolet absorption spectrophotometry. Using this procedure, the molar proportions of the Nm constituents of wheat embryo, yeast, and Escherichia coli tRNA have been determined. The close correspondence between the values measured by venom hydrolysis and those obtained by analysis of alkali-stable dinucleotide (Nm-Np) sequences attests to the validity of the venom assay, and further indicates that alkali-stable sequences larger than dinucleotides are not present in significant amounts in the tRNA of the above three organisms.During the present investigation, several ultraviolet-absorbing components, not immediately identifiable as ribose-methylated nucleotides, were isolated along with the expected O2′-methylnucleoside 5′-phosphates. Preliminary characterization of one of these compounds suggests that it is a derivative of a novel nucleoside, O2′-methyl-5-carboxymethyluridine (cm5Um). Venom hydrolysis of yeast tRNA liberates the 5′-nucleotide of cm5Um in the form of a carboxyl-blocked derivative (pU-2). During alkaline hydrolysis of yeast tRNA, the blocking group in U-2 is labilized and cm5Um is released as part of an alkali-stable dinucleotide, cm5Um-Ap. The proportion of pU-2 in venom hydrolysates of yeast tRNA (0.02 mol%, the same as the content of cm5Um-Ap in alkaline hydrolysates) suggests that O2′-methyl-5-carboxymethyluridine may be confined to a single isoaccepting species of tRNA in yeast.In an allied study, reinvestigation of the alkali-stable dinucleotide sequences of baker's yeast tRNA has confirmed previous results concerning the sequence distribution of O2′-methylribose in yeast tRNA (Gray, M. W. &Lane, B. G. (1967) Biochim. Biophys. Acta 134, 243–257).

END GROUP AND SEDIMENTATION DATA ON FRAGMENTED HIGH MOLECULAR WEIGHT RIBONUCLEATES

1963 ◽  
Vol 41 (1) ◽  
pp. 1927-1941 ◽  
Author(s):  
B. G. Lane ◽  
J. Diemer ◽  
C. A. Blashko
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Snake Venom ◽  
Group Analysis ◽  
Sedimentation Coefficient ◽  
Snake Venom Phosphodiesterase ◽  
Ehrlich Ascites ◽  
Spontaneous Hydrolysis ◽  
Hydrolysis Of ◽  
End Group

A method for end group analysis of ribonucleate preparations using purified snake venom phosphodiesterase is described. Unusual difficulties encountered with the method are discussed. The technique is useful for detection of end groups resulting from enzymic and chemical fragmentation of high molecular weight ribonucleates. Preliminary studies indicate that the method has limited usefulness because of a spontaneous hydrolysis of ribonucleates which occurs under the conditions which are optimal for hydrolysis with snake venom phosphodiesterase (pH 9, in the presence of magnesium). Physicochemical studies have shown that the pronounced dependence of sedimentation coefficient on ionic strength which has been reported by other investigators is also observed with fragmented high molecular weight ribonucleates and with 16S + 24S ribonucleates of Ehrlich ascites cells. The changes of sedimentation rate are associated with configurational and aggregation effects.

END GROUP AND SEDIMENTATION DATA ON FRAGMENTED HIGH MOLECULAR WEIGHT RIBONUCLEATES

1963 ◽  
Vol 41 (9) ◽  
pp. 1927-1941 ◽  
Author(s):  
B. G. Lane ◽  
J. Diemer ◽  
C. A. Blashko
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Snake Venom ◽  
Group Analysis ◽  
Sedimentation Coefficient ◽  
Snake Venom Phosphodiesterase ◽  
Ehrlich Ascites ◽  
Spontaneous Hydrolysis ◽  
Hydrolysis Of ◽  
End Group

A method for end group analysis of ribonucleate preparations using purified snake venom phosphodiesterase is described. Unusual difficulties encountered with the method are discussed. The technique is useful for detection of end groups resulting from enzymic and chemical fragmentation of high molecular weight ribonucleates. Preliminary studies indicate that the method has limited usefulness because of a spontaneous hydrolysis of ribonucleates which occurs under the conditions which are optimal for hydrolysis with snake venom phosphodiesterase (pH 9, in the presence of magnesium). Physicochemical studies have shown that the pronounced dependence of sedimentation coefficient on ionic strength which has been reported by other investigators is also observed with fragmented high molecular weight ribonucleates and with 16S + 24S ribonucleates of Ehrlich ascites cells. The changes of sedimentation rate are associated with configurational and aggregation effects.

The chain termini of polynucleotides formed by limited enzymic fragmentation of wheat embryo ribosomal RNA. Part 2. Studies of a snake venom ribonuclease and pancreas ribonuclease

1968 ◽  
Vol 46 (1) ◽  
pp. 93-107 ◽  
Author(s):  
B. D. McLennan ◽  
B. G. Lane
Keyword(s):  
Snake Venom ◽  
Ribosomal Rna ◽  
Mode Of Action ◽  
Ph Dependence ◽  
Wheat Embryo ◽  
Analytical Technique ◽  
Phosphodiester Bonds ◽  
Acetone Fractionation ◽  
High Degree ◽  
End Group

A snake venom ribonuclease, similar to the classical pancreas ribonuclease, can be freed of other known nucleolytic activities by acid treatment, or by acetone-fractionation of Russell viper venom. Utilizing an end-group analytical technique in conjunction with oligonucleotide analyses of both partial and complete digests of ribonucleates, it has been possible to broadly characterize the mode of action of the snake venom ribonuclease, even though it is present in such small amount in venom that extensive purification was not attempted. The venom ribonuclease parallels pancreas ribonuclease in its acid stability, pH dependence, and high degree of preferential specificity toward PypA bonds in ribonucleate chains. Studies of the limited fragmentation of ribosomal and soluble ribonucleates, as well as dinucleoside phosphates, have shown that there are subtle differences between the venom and pancreas ribonucleases. The results of the investigation suggest that the venom and pancreas ribonucleases can be useful as a means of introducing a limited number of preferential scissions into ribonucleate chains at PypA internucleoside phosphodiester bonds. Incidental to the principal investigations dealing with the mode of action of the venom and pancreas ribonucleases on ribonucleates, certain features pertaining to the nucleotide sequences in ribosomal and soluble ribonucleates from wheat embryo have been noted.

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