Rusty J. Mans

Conservation of the primary structure at the 3′ end of 18S rRNA from eucaryotic cells

DNA sequencing methods have been used to determine a sequence of about 20 nucleotides at the 3 termini of various 18S (small ribosomal subunit) RNA molecules. Polyadenylated rRNA was first synthesized using the enzyme ATP:polynucleotidyl transferase from mainze. Then in the presence of an oligonucleotide primer uniquely complementary to the end of each adenylated rRNA, a cDNA copy was produced using AMV reverse transcriptase. In every case, the cDNA transcript was of finite size, which we ascribe to the appearance of an oligonucleotide containing m62A near the 3 end of the 18S rRNAs. Sequences at the 3 termini of 18S rRNA molecules from the four eucaryotic species examined here (mouse, silk worm, wheat embryo and slime mold) are highly conserved. They also exhibit strong homology to the 3 end of E. coli 16S rRNA. Two important differences, however, are apparent. First, the 16S sequence CCUCC, implicated in mRNA binding by E. coli ribosomes, is absent from each eucaryotic rRNA sequence. Second, a purine-rich region which exhibits extensive complementarity to the 5 noncoding regions of many eucaryotic mRNAs appears consistently.

In vitro synthesis of DNA complementary to polyadenylated histone messenger RNA

Summary The messenger RNAs for histone polypeptides were isolated from S phase HeLa S3 cells and adenylic acid residues were enzymatically added to the 3′-OH end of the molecules with a eukaryotic ATP: polynucleotidylexotransferase. The polyadenylated histone messenger RNAs were then used as templates for the synthesis of complementary DNAs by RNA-dependent DNA polymerase from Rous sarcoma virus.

Cell cycle stage-specific transcription of histone genes.

Abstract DNA complementary to histone messenger RNAs was utilized to assay the in vitro transcripts from chromatin of G1 and S phase HeLa S3 cells for the presence of histone specific sequences. The complementary DNA was prepared by transcribing, with an RNA-dependent DNA polymerase, histone messenger RNAs isolated from the polyribosomes of S phase HeLa S3 cells to which adenylic acid residues had been enzymatically added. While the RNA transcripts of S phase chromatin contained histone specific sequences, such sequences were not detected in the RNA transcripts from G1 chromatin. These results suggest that transcription of the genes for histone polypeptides is restricted to the S phase of the cell cycle.

Transfer RNA-primed oligoadenylate synthesis in maize seedlings: I. Requirements of the reaction and nature of the product with crude enzyme☆

Abstract 1. 1. An enzyme, isolated from corn seedlings, catalyzes the addition of AMP residues to tRNAs upon incubation with ATP, Mn2+, dithiotreitol and tRNA. 2. 2. The AMP residues are in 3′,5′-phosphodiester linkage and the oligonucleotide is covalently linked to the 3′-terminus of the primer tRNA. 3. 3. The enzyme is similar to poly A polymerases isolated from rat liver and Escherichia coli; however, the corn enzyme exhibits specificity for tRNAs.

Transfer RNA-primed oligoadenylate synthesis in maize seedlings. III. Deoxyoligonucleotide primers

Abstract Single stranded DNA can replace tRNA as the nucleic acid component required for ATP incorporation by the adenylating enzyme from maize seedlings. Preliminary data suggest that deoxyoligonucleotides serve as primers for addition of AMP moieties to 3′ hydroxyl termini. The strict requirement for manganese is abrogated by desalting the enzyme.

Transfer RNA-primed oligoadenylate synthesis in maize seedlings: II. Primer, substrate and metal specificities and size of product

Abstract 1. The oligoadenylating enzyme, partially purified from corn seedlings, catalyzes the synthesis of polyadenylate linked to the 3′-hydroxyl of the terminal nucleoside of an RNA primer. 2. rRNA, a mixture of tRNAs, methionyl-tRNA or tRNA lacking a terminal adenosine satisfy the primer requirement. The enzyme is specific for ATP and Mn 2+ . 3. The maize enzyme is analogous to the adenylate-incorporating enzyme of rat liver. A relationship between the corn polyadenylic acid polymerase and RNA polymerase is postulated.

Addition of polyadenylic acid to RNA by ATP: Polynucleotidylexotransferase partially purified from HeLa cells

Abstract An enzyme that catalyzes the addition of polyadenylic acid to several RNAs was partially resolved by salt precipitation and DEAE-cellulose chromatography from the cytoplasmic component of HeLa cells. ATP and manganese are required for the sequential addition of AMP moieties to lengthen RNA primers. The presence of endogenous primer and endonucleolytic activity associated with the enzyme are indicated.

Selection of repeated sequences of homologous and heterologous DNA during invitro transcription by maize RNA polymerase

Abstract RNA synthesized in vitro by maize RNA polymerase II arises in part from repeated DNA sequences, since significant hybridization to the parent DNA occurs with low concentrations of RNA and DNA. Over three times as much “repeated sequence” RNA is transcribed from maize as from calf thymus DNA.

Transcription of circular, single-stranded DNA by maize RNA polymerase II

Selective initiation of RNA chains on DNA templates by eukaryotic RNA polymerases may underlie cellular differentiation [ 1 ] and responses to environmental stimuli. Initiation of RNAs with ATP and GTP by eukaryotic RNA polymerases was inferred from the incorporation of [Y-~~P] nucleosidetriphosphates into acid-insoluble material [2] but the products were not rigorously identified (other than viral [3] ). With maize RNA polymerase II, we could demonstrate incorporation of [Y-~~P] ATP into products accumulated on several templates but little was DNA-dependent (unpublished). Maize polymerase selectively transcribes maize DNA rather than a heterologous DNA when offered in the same reaction mixture [4] ; demonstrating a measure of selective binding and chain initiation. We turned to a homogeneous and defined DNA to determine which template properties lead to polymerase binding and RNA chain initiation. Under certain conditions, template termini are known to serve as non-specific sites of chain initiation [S] . Mandel and Chambon [6], however, have demonstrated in vitro transcription of SV40-FI DNA by calf thymus RNA polymerases A and B, indicating that unnicked templates can be transcribed by these enzymes. The use of circular @Xl 74 DNA precludes termini as potential sites of initiation, unless generated by an endonuclease in the RNA polymerase preparation. In addition, nicking of

Rifampicin inhibition of poly(dT)·Poly (A)-primed poly (A) synthesis by HeLa AMP polynucleotidylexotransferase

X174 DNA does not significantly alter the physical structure of the DNA as does nicking of super-helical SV40-FL We report here that single-