The priming and lyophilized. This article must therefore be hereby marked Alu, Bam, Hae, and Hpa, restriction nucleases isolated from Athro- Downloaded by guest on June 7, "advertisement" in accordance with 18 U. USA 75 0. In preparative ex- periments the fractions sedimenting with the template were saved for gel electrophoresis.
The plate was washed in methanol, dried, and autoradiographed. A transcript was formed in the presence of nucleoside triphosphates Fig.
The synthesis decreased after 20 min but did not level off com- pletely. Low concentration of nucleoside triphosphates reduced the RNA synthesis. Gel electrophoresis of ori-RNA. RNA synthesis was with was synthesized on the genome. The amount of RNA synthesized decreased as binding protein I. The length of the ori-RNA was calculated from its Downloaded by guest on June 7, the salt concentration increased. As will be discussed below, the mobility relative to ssDNA fragments of 20 and 42 nucleotides.
Biochemistry: Geider et al. ELo 4. The bands of the ori-RNA Fig. Both properties suggest that the RNA is isolated as a stable hybrid. Analysis of the ori-RNA. A dominant band was visualized in a size class of 30 nucleotides. The nine spots indicate a unique se- quence for the ori-RNA. Their composition was further in-.
Table 1. The samples were 1 3'-OH oligonucleotide C-G Nearest neighbor The spots from the chromatogram Fig. U-G-G-OH hydrolysis. This spot localizes another end at position The numbers indicate the site of the largest of the four DNA fragments marked by asterisks in Fig.
The two restriction fragments at each cleavage site are indicated by capital letters. Label fragment 6. Spot 8 has the nucleotide-long highly base-paired structure. We the composition of spot 9, but the y-terminal phosphate was lost suggest that after addition of ribonucleoside triphosphates RNA during preparation.
After incorporation of a short a to nucleotide-long RNA. Two distortions in the base-paired se- continued. The product was restricted in parallel experiments quence facilitate this process and favor the hybridization of the with the four restriction enzymes Fig. Unspecifically RNA towards the hairpin structure. DNA binding protein I primed DNA molecules produced some background synthesis immediately complexes the opposite chain of the opening helix that gave the normal restriction fragments.
In addition, four and finally runs into the loop on top of the hairpin. The relative the termination of the ori-RNA by interfering with the move- intensities of these bands varied similarly in the three experi- ment of the polymerase, because RNA polymerase cannot ments, indicating that they were in each experiment derived displace DNA binding protein I on a single-stranded template from the same set of four closely related primer RNA chains.
Geider, unpublished data. The stop of transcription does The distance from the cleavage sites Fig. This site overlaps with the polymerase or the binding protein proceeds faster.
A variable ori-DNA. The polymerase begins de- one site Fig. This is the same site as identified by restriction oxynucleotide polymerization by extension of the RNA end. The ori-RNA starts at an exact point, DNA binding protein I then favors the action of the DNA and the end is reached after more than 25 nucleotides, which polymerase 2 and allows replication downstream from this is near the loop of the hairpin structure used for RNA synthesis hairpin region.
After almost one cycle of replication the poly- Fig. In this lI- OH Spot no. Alignment of the ori-RNA fragments. Tabak, H. Geider, unpublished results. Horiuchi, K. Table 1. The samples were 1 3'-OH oligonucleotide C-G Nearest neighbor The spots from the chromatogram Fig.
U-G-G-OH hydrolysis. This spot localizes another end at position The numbers indicate the site of the largest of the four DNA fragments marked by asterisks in Fig.
The two restriction fragments at each cleavage site are indicated by capital letters. Label fragment 6. Spot 8 has the nucleotide-long highly base-paired structure.
We the composition of spot 9, but the y-terminal phosphate was lost suggest that after addition of ribonucleoside triphosphates RNA during preparation. After incorporation of a short a to nucleotide-long RNA. Two distortions in the base-paired se- continued. The product was restricted in parallel experiments quence facilitate this process and favor the hybridization of the with the four restriction enzymes Fig.
Unspecifically RNA towards the hairpin structure. DNA binding protein I primed DNA molecules produced some background synthesis immediately complexes the opposite chain of the opening helix that gave the normal restriction fragments. In addition, four and finally runs into the loop on top of the hairpin. The relative the termination of the ori-RNA by interfering with the move- intensities of these bands varied similarly in the three experi- ment of the polymerase, because RNA polymerase cannot ments, indicating that they were in each experiment derived displace DNA binding protein I on a single-stranded template from the same set of four closely related primer RNA chains.
Geider, unpublished data. The stop of transcription does The distance from the cleavage sites Fig. This site overlaps with the polymerase or the binding protein proceeds faster. A variable ori-DNA. The polymerase begins de- one site Fig. This is the same site as identified by restriction oxynucleotide polymerization by extension of the RNA end.
The ori-RNA starts at an exact point, DNA binding protein I then favors the action of the DNA and the end is reached after more than 25 nucleotides, which polymerase 2 and allows replication downstream from this is near the loop of the hairpin structure used for RNA synthesis hairpin region. After almost one cycle of replication the poly- Fig. In this lI- OH Spot no. Alignment of the ori-RNA fragments. Tabak, H. Geider, unpublished results. Horiuchi, K. USA We thank E. Piaskowski for skillful technical assistance.
This work 73, Geider, K. Reichard, P. Wickner, W. USA 71, USA 69, Bouche, J. Rowen, S. Brutlag, D. Berthold, B. USA 68, Sigal, N. Burgess, R. McHenry, C. Mirzabekov, A. Schaller, H. Heyden, B. USA 73, Gray, C. USA Proc.
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