Poly polymerase materials

Adenosine triphosphate, when oxidized to its dialdehyde with periodate and condensed with Sepharose-adipic (or sebacic) acid hydrazide, has been used to isolate poly(A) polymerase, and also myosin and its fragments from different sources. A poly(A)-linked resin has been prepared by condensing 4,4 -diaminodiphenyl-methane with periodate-oxidized starch, reducing the resulting Schiff bases with borohydride, diazotizing, and then coupling with the polynucleotide. The resulting material may be used to isolate poly(U) sequences. 

A dipstick probe can be used to identify organisms in clinical or environmental specimens. It consists of adding a capture probe (containing a sequence of poly-dA or -dT bases that identifies it as a capture probe) coupled to dioxygenin as a reporter. Following destruction of unhybridized probe material, a dipstick that can hybridize with the capture probe base sequence is added. After all steps are completed, the presence of the capture probe is positive for the organism of interest. The theoretical sensitivity of a probe is < 1 pg of nucleic acid or 10 cells. However, by use of polymerase chain reaction (PCR), the DNA of a single cell can be amplified several millionfold. 

In the late seventies it was reported that several chromatin proteins could be 2 dADP-ribosylated following incubation of a erode preparation of nuclei with radiolabeled 2 dNAD+ (17, 18). These observations have since been interpreted to indicate that 2 dNAD+ is a substrate for poly(ADP-ribose) polymerase (19). However, pure poly(ADP-ribose) polymerase does not utilize 2 dNAD as a substrate vide supra. Thus, the incorporation of radiolabeled 2 dNAD+ into acid insoluble material with crude rat liver nuclei may have resulted from either (i) the non-enzymatic ADP-ribosylation of chromatin proteins (20, 21) or (ii) the activity of a mono(ADP-ribosyl) transferase (4). 

Fig. 3. Immunoblots of subcellular fractions of CHO and CALU-1 cells. In summary, cells (5 X 10 ) peimeabilized with 1% NP-40 (NP) were treated at 0° C with 250 pg/ml of DNase I and ase A for 2 hr (RD), extracted for 1 hr with 2M NaCl buffer (S), and centrifuged to yield insoluble material (P). The different subcellular fractions (NP,RD,S,P), re senting 100% of cellular protein content, were dissolved in a 4 M urea SDS sample buffer and subjected to western blot analyses. Poly(ADP-ribose) polymerase from CHO and CALU-1 cells were revealed in these cases with C-2-10 and C-1-9 antibodies respectively. Purified calf thymus enzyme (E) is used as reference.

Escherichia coli RNA-polymerase hole- and core enzymes were 95% pure by sodium dodecyl sulfate (SDS) gel electrophoresis. Enzyme activity was measured as the amount of [ C]AMP or [ C]UMP incorporated into acid-insoluble material after a 10-min incubation at 37°. The assay mixture contained in 0.1 ml 40 mM Tris-chloride at pH 8.0, 8 mM MgCl2, 5 mAf dithioerythritol, 0.2 Azoo unit of poly[d(AT)], 0.05 M KCl, 1 mAf ATP, and 1 mAf [ C]UTP. For kinetic studies, a fixed concentration of ATP (0.4 mAf) was used and the concentration of [ C]UTP was varied. Enzyme activity was measured as the amount of [ C]UTP incorporated into acid-insoluble material after 5 min. 

Polysaccharides are available from plants (starch, cellulose, alginate), animals (chitin), fungi (pullulan) and bacteria (dextran, emulsan, pectin). For a sununary on industrial polysaccharides see Stivala et al. [4, S]. Proteins are produced by all living species in order to maintain their metabolic functions, but from a materials point of view it is the fibrous proteins from plants (soy), animals (wool, silk) [6] and bacteria (polyglutamic acid) that are exploited. Lignin, a polyphenolic compound, [7] and natural rubber, a polyisoprene, [8] are synthesized by plants. Finally, poly(hydroxyalkanoates) are synthesized naturally exclusively by bacteria [9]. Bacterial polymerase genes have also been successfully transferred to plants [10]. 

Replication of synthetic homopolyribonucleotides has been studied by Friedemann Schneider and coworkers (Schneider et al., 1979 Heinrichs and Schneider, 1980). They studied RNA-synthesis at poly(A)-poly(U) templates by the unspecific RNA polymerase from E. coli in a stirred flow reactor. In general this reaction follows an overall autokatalytic kinetics. In the flow reactor the recycling process is replaced by an influx of A and an outflux of the material in the reactor. It is worth noticing that critical slowing down has been observed experimentally in this system. 

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