RNA-ases

It should be pointed out that when using ethidium bromide the sensitivity of the assays varies depending on the physical state of the nucleic acids (see Table I). Ethidium does not discriminate between RNA and DNA, although dyes are available which bind DNA exclusively, so the relative amounts of each may be determined by taking two sets of measurements. Alternatively, nucleases (DNA-ase or RNA-ase) can be used to exclusively remove one or the other in a mixture. Nucleic acids from different sources (see Table II) also show a variation in sensitivity, and the fluorescence assay lacks the selectivity of the hybridization technique. Nevertheless, for rapid screening or quality-control applications the fluorescence assay is still the method of choice. 

CRITICAL ASSESSMENT OF THE METHOD Since differential expression analysis means to compare the quantities of RNA species in two samples, every step during sample preparation has to be highly reproducible. In order to maximize reproducibility, complete total RNA extraction in a one step procedure is recommended. Care must always be taken when working with RNA, to avoid contamination with RNA ases, which may result in RNA degradation. 

A Anaerobic adaptation was obtained by flushing the cells with argon. At indicated time points, samples were taken to measure the in vitro Fk-ase activity of C. reinhardtii (box), S. obliquus (circle), C. fusca (triangle), C. moewusii (diamond). While the activities of both Scenedesmus species are comparative low, the in vitro H -production rate of anaerobically induced C. moewusii cultures is 2 times higher than the activity of induced C. reinhardtii cultures. B Northern blots with equal amounts of total RNA isolated from an anaerobically adapted culture (2 h) and an uninduced reference culture (0 h) of C. moewusii. The upper blot was incubated with a RNA sample of the 3 UTR of the hydA 1 cDNA, while the lower blot was incubated with a RNA sample generated of the cDNA from the constitutively expressed sedoheptulose 1,7-bisphosphatasegene. 

Gribskov, M. (1992). Translational initiation factors IF-1 and eIF-2 alpha share an RNA-binding motif with prokaryotic ribosomal protein SI and polynucleotide phosphoryl-ase. Gene 119, 107-111. 

The microsomal fraction was first obtained by Claude in 1943. In addition to lipid in the fraction, he noted the presence of RNA-rich granules, consistent with reports from Brachet that cytoplasm stained for RNA by the methyl-green/pyronin procedure. Glucose-6-phos-phatase was a prominent enzyme when the fraction was prepared from liver. Since density gradient sedimentation showed G-6-P-ase was absent from mitochondria and lysosomes, it was used as a marker for liver microsomes. 

DNA ligase (NAD+) [EC 6.5.1.2] (also referred to as polydeoxyribonucleotide synthase (NAD+), polynucleotide ligase (NAD+), DNA repair enzyme, and DNA join-ase) catalyzes the reaction of NAD+ with (deoxyribo-nucleotide) and (deoxyribonucleotide) to produce AMP, nicotinamide nucleotide, and (deoxyribonucleo-tide)( +m). This forms a phosphodiester at the site of a single-strand break in duplex DNA. RNA can also act as substrate to some extent. 

These reactions are the easiest to tackle, since they require only one phosphoryl oxygen to be substituted in both the substrate and the product. The classic example of this experiment is the first step in the hydrolysis of RNA catalyzed by bovine pancreatic ribonuclease. As discussed in detail in Chapter 16, ribonucle-ase catalyzes the hydrolysis of RNA by a two-step reaction in which a cyclic intermediate is formed. The stereochemistry of the first step (cyclization) (equation 8.35),... 

Zinc is involved in many biochemical functions. Several zinc metal-loenzymes have been recognized in the past decade. Zinc is required for each step of cell cycle in microoragnisms and is essential for DNA synthesis. Thymidine kinase, DNA-dependent RNA polymerase, DNA-polymer-ase from various sources, and RNA-dependent DNA polymerase from viruses have been shown to be zinc-dependent enzymes. Zinc also regulates the activity of RNase, thus the catabolism of RNA appears to be zinc dependent. The effect of zinc on protein synthesis may be attributable to its vital role in nucleic acid metabolism. 

The controversy on the existence of in vivo Diels-Alder reactions cannot be put to rest here, but the numerous examples of natural products containing cyclohexene groups and the catalytic effectivity of biological surroundings support the idea of in vivo Diels-Alder reactions. Apart from cell-free extracts, RNA-based mixtures of metals also show catalytic activity and it was demonstrated that this catalyst system can be quite effective as an artificial Diels-Alder-ase . We will show that water, the prime solvent of biosynthesis, also catalyses [4 -+- 2]-cycloadditions. Considering that biosyntheses are often of exceptional selectivity, it is clear that understanding biomimetic transfonna-tions in water as the solvent is an important goal of modem chemistry. The possibilities offered by and the reasons for Diels-Alder catalysis in water will be the main topic of this chapter. 

Transcription, the synthesis of RNA from a DNA template, is carried out by RNA polymerases (Fig. 14.2). Like DNA polyma-ases, RNA polymerases catalyze the formation of ester bonds between nucleotides that base-pair with the complementary nucleotides on the DNA template. Unlike DNA polymerases, RNA polymerases can initiate the synthesis of new chains in the absence of primers. They also lack the 3 to 5 exonuclease activity found in DNA polymerases. A strand of DNA serves as the template for RNA synthesis and is copied in the 3 to 5 direction. Synthesis of the new RNA molecule occurs in the 5 to 3 direction. The ribonucleoside triphosphates ATP, GTP, CTP, and UTP serve as the precursors. Each nucleotide base sequentially pairs with the complementary deoxyribonucleotide base on the DNA template (A, G, C, and U pair with T, C, G and A, respectively). The polymerase forms an ester bond between the a-phos-phate on the ribose 5 -hydroxyl of the nucleotide precursor and the ribose 3 -hydroxyl at the end of the growing RNA chain. The cleavage of a high-energy phosphate bond in the nucleotide triphosphate and release of pyrophosphate (from the (3 and y phosphates) provides the energy for this polymerization reaction. Subsequent cleavage of the pyrophosphate by a pyrophosphatase also helps to drive the polymerization reaction forward by removing a product. 

Both the inhibition and poisoning of topoisomerases are deleterious to cells. The collision of a transcription complex or a replication fork against a topoisomer-ase-associated DNA break interrupts RNA or DNA synthesis, and can lead to real (nontopoisomerase-bound) double-strand breaks and to gene translocations, which can trigger apoptosis and/or cancer (Li and Liu 2001). 

M-MulV Reverse transcriptase isolated from moloney murine leukemia virus. This enzyme is also characterized by RNA-dependent DNA polymerase, DNA-dependent DNA polymerase and weak RN ase H activities but lacks the 3 -5 exonuclease activity. M -MulV reverse transcriptase is able to synthesize full-length cDNA fragments from longmRNA.Thetemperaturefortheoptimalenzymeactivity is 37 °C. Becauseofthe weak RNase H activities of the M-MulV reverse transcriptase, it is more suitable for the synthesis of long cDNA fragments. 

Functionally, PCs are the morphological expression of the DNA-rich compartment of the nucleolus and contain most of the DNA-associated components, such as transcription-related proteins of the RNA polymerase I enzyme complex (Scheer and Rose, 1984) and the transcription factor UBF/NOR-90 (Chan et ai, 1991 Rodrigo et al., 1992 Roussel et ai, 1993). Besides these, DNA topoisomer-ase I has also been reported to be in PCs (Raska et al., 1990b) as well as throughout the nucleoplasm. Thus, any protein localized to PCs is probably associated with the ribosomal DNA in replicative or transcription-related events. Thus far, antibodies to RNA polymerase I have been used as the only specific marker for PCs (Fig. 5, a and b) because other proteins in the PCs also exist in other structures as well (see Table I), and Ag-NOR staining labels both PCs and the DFC (Ploton et al, 1986). 

RNA polymerase has now been purified in various laboratories from various mammalian sources [188], and RNA polymerase of eukaryotic nuclei always exists in multiple forms that can be distinguished by the cation requirement, their sensitivity to a-amanitin, and their ability to react with specific templates. One enzyme (polymerase I) is found in the nucleolus, the others in the nucleoplasm. All mammalian ribonucle-ases are complex proteins formed of several subunits. Three types of RNA polymerases have been purified from ascites tumor cells by chromatography on car-boxymethyl-cellulose. Two are nucleolar and one is nucleoplasmic. Protein factors of unknown nature that stimulate all three enzymes have been found in calf thymus, rat liver, and ascites cells. These factors can be separated into two classes heat stable and heat labile. Both types stimulate the activity of the Novikoff RNA polymerase several-fold, but only with native DNA as templates. The factors have no effect on E. coli RNA polymerase [266-267]. For further information, refer to the review of Jacobs [189]. 

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