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C G Merkel

Publications and source records attributed to C G Merkel.

7 recordsLinked to original sources
PubMed1977

Methylated nucleosides in globin mRNA from mouse nucleated erythroid cells.

Poly(A)-containing mRNAs labeled with [methyl-3H]methionine were isolated from nucleated erythroid cells obtained from the spleens of anemic mice. The RNAs were further separated into non-globin poly(A)-containing RNAs and highly purified globin mRNA by globin cDNA-cellulose affinity chromatography. DEAE-Sephadex column chromatography of the T2 ribonuclease digestion products of the cDNA-purified globin mRNA fraction yielded methylated resistant fragments with charges of -4.7 (Cap 1) and -5.3 (Cap 2). Digestion of the non-globin RNA fraction revealed a similar pattern with the addition of a methylated mononucleotide identified as 6-methyladenosine at -2 charges. Alkaline phosphatase treatment of the T2 resistant fragments reduced their charges by approximately 2, which is consistent with the removal of one terminal phosphate. Treatment of the globin T2 and alkaline phosphatase-resistant fragments withpenicillium P1 nuclease and alkaline phosphatase yielded a P1-resistant core structure in both fragments. In addition to the core, 2'-O-methylcytidine (Cm) was released from the more negatively charged globin fragment. The P1-resistant cores of the cap structures eluted from DEAE-Sephadex with the known standard m2G5'ppp5'Am and were found to be pyrophosphatase-sensitive establishing a 5'-5'-triphosphate linkage. The pyrophosphatase and alkaline phosphatase digestion products of the globin Cap 1 and Cap 2 core structures were analyzed by high voltage electrophoresis and paper chromatography and found to be 7-methyiguanosine (m7G) and the dimethylated nucleoside 6-methyl-2'-O-methyladenosine (N6mAm). A small amount of the singularly methylated adenosine, 2'-O-methyladenosine (Am) was also observed. The predominant sequences of the methylated nucleosides in the globin cap structures are therefore m7G5'ppp5'N6mAm and m7G5'ppp5'N6mAmpCm.

Animals↗
PubMed1976

Shortening of the poly(A) region of mouse globin messenger RNA.

Nucleated erythroid cells isolated from the spleens of anemic mice were used to investigate the processing of the polyadenylic acid region of globin mRNA. Cells were labeled in media containing [3H] adenosine and transferred to media containing no radioactive precursor and incubated further in the presence or absence of actinomycin D. After various times following the transfer of the cells, globin mRNA was isolated using a combination of oligo(dT)-cellulose affinity chromatography, sucrose density centrifugation, and globin cDNA (the complementary DNA copy of globin mRNA)-cellulose affinity chromatography. The size of the poly(A) region was determined by polyacrylamide gel electrophoresis of the T1 and pancreatic RNase-resistant fragments. The prelabeled poly(A) region which initially comprises approximately 150 adenylate residues was found to become shorter with time, both in cells incubated in medium containing no radioactive precursor and in the presence of actinomycin D. After 9 h of incubation in the presence of actinomycin D, two major size classes of poly(A) were observed, one containing 35 to 45 adenylic acid residues and the other containing 55 to 65 residues. These two size classes are similar to those found in circulating reticulocytes suggesting that the poly(A) shortening observed in these cell incubation studies is similar to that which occurs in vivo. Two protein synthesis inhibitors, emetine and cycloheximide, were investigated with respect to their effect on poly(A) shortening. Neither drug inhibited the shortening of the poly(A) region of globin mRNA, suggesting that protein synthesis is not required for this process to occur.

Animals↗
PubMed1975

Size of the polyadenylic acid region of newly synthesized globin messenger ribonucleic acid.

The size of the polyadenylic acid region of newly synthesized globin mRNA was determined on mRNA isolated from nucleated erythroid spleen cells of anemic mice. The globin mRNA was purified by a combination of affinity chromatography on oligodeoxythymidylate-cellulose (oligo-(dT)-cellulose) and sucrose density gradient centrifugation. The purified RNA was shown to be globin mRNA by virtue of its ability to direct the synthesis of mouse alpha- and beta-globin chains in a cell-free system and by the presence of two bands migrating identically with authentic mouse alpha- and beta-globin mRNA when subjected to electrophoresis in polyacrylamide gels in the presence of formamide. When labeled for 1 hour with [3H]adenosine, the newly synthesized radioactive mRNA also migrated as two bands in these gels but they moved slower than the main bands suggesting that they have higher molecular weights. The polyadenylic acid region of the mRNA was isolated from the T1 and pancreatic RNase digestion mixture by acrylamide sodium dodecyl sulfate gel electrophoresis. The polyadenylic acid region was found to contain approximately 150 adenylate residues. As it is known that globin mRNA isolated from reticulocytes contains only 40 to 60 residues, it follows that at least 150 adenylic acid residues are added to the globin mRNA soon after its synthesis and that some of these are removed during the subsequent maturation of the erythroid cell.

Adenine Nucleotides↗