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Minus strand synthesis begins after the completion of ..
Figure 1. Synthesis Kit by oligo(dT) primer. The synthesized cDNA from human placenta was then used to amplify different gene regions by quantitative PCR using the All-in-One qPCR Mix (GeneCopoeia Catalog No. AOPR-0200). The positive amplification result of MACF1 indicates that up to 13 kb RNA sequence was reversed transcribed.
So why do we care about genomic DNA contamination? This is because genomic DNA found in RNA preparations can act as an effective template during PCR, resulting in undesirable false positive signals unrelated to mRNA. Strategies such as cross-intron primer design may still not be able to avoid amplification from the contaminating genomic DNA due to presence of large number of processed pseudogenes in the mammalian genomes.
of plus- or minus-strand RNA for ..
RNase H makes bothspecific and nonspecific cleavages; specific cleavages are used togenerate and remove the polypurine tract primer used for plus-strand DNAsynthesis and to remove the tRNA primer used for minus-strand DNAsynthesis.
The ends of the linear DNA form of the HIV-1 genome are defined by thespecific RNase H cleavages that remove the plus- and minus-strandprimers; these ends can be joined to form two-long-terminal repeatcircles.
of minus-strand synthesis in vitro ..
The RNA-dependent RNA polymerase of bacteriophage Qb was first isolated in 1965 by Haruna and Spiegelman, who named it Qb replicase (1). Today, this enzyme still represents the prototype of RNA virus-replicating polymerases, and it has remained unique in that it allows the specific replication and amplification of an infectious viral RNA by a complex of soluble, stable, and defined protein components in vitro. Early studies had established that the viral RNA genome (the plus strand) was replicated by primer-independent end-to-end RNA synthesis in the 5′ to 3′ direction. A free, single-stranded complementary strand (minus strand) is synthesized as an intermediary product and serves as a template for the synthesis of single-stranded plus strands, whereas double-stranded RNA is devoid of any template activity (2). Investigations with Qb replicase have resulted over the years in a surprising number of novel concepts of more general relevance, such as: (1) specific template recognition by a polymerase (1); (2) recruitment of host proteins as subunits of a viral polymerase (3, 4); (3) role of a viral polymerase in the temporal control of protein biosynthesis (2); (4) an internal RNA binding site as an enhancer of synthesis initiation (5); (5) activation of an RNA template by a host factor acting as an RNA molecular chaperone (6); (6) secondary and tertiary structure as determinants of template recognition (7, 8); (7) role of secondary structure in elongation of RNA synthesis (9, 10); (8) sequence evolution in vitro (11) and the quasispecies concept (12); (9) de novo synthesis of replicatable RNA (13); (10) site-directed mutagenesis (14); (11) use of small RNA templates as vectors for foreign sequences (15); and (12) development of such systems for diagnostic purposes (16). Most of these aspects will be discussed here.
For an RNA to be replicated and amplified by replicase, both the RNA itself and its complementary strand must be efficient templates. This is the case for Qb RNA and the Qb minus strand in the presence of the holoenzyme and host factor. The core enzyme without the host factor is sufficient to copy the minus-strand RNA into a plus strand, but cannot by itself use the latter as an efficient template. However, the core enzyme is efficient for replicating and amplifying a family of small RNAs, collectively called 6 S RNA, that arise in vitro (and possibly also in vivo) in the absence of added template RNA, either by uninstructed de novo synthesis or through elongation and recombination of small RNA fragment contaminants (see below). RNAs of closely related phages (eg, SP) have partial activity (23), but more distant phage RNAs (eg, MS2), like other viral and cellular RNAs, are inactive. Synthetic templates like poly(C) and mixed polyribonucleotides rich in C function as templates for the synthesis of a complementary strand, which however remains paired with the template and is itself not active as a template.
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(minus strand) is synthesized as ..
Recently, the SELEX technique was used to generate two families of short RNA ligands that bound strongly to replicase holoenzyme and were active templates (30). One contained a pseudoknot structure rich in A and C residues in the unpaired loops and was found to crosslink to S1 protein. The other contained a polypyrimidine tract and crosslinked to EF-Tu (the g subunit). At present, it appears difficult to integrate these findings into a common picture with those from the Qb plus and minus strand.
Ribosomes - Protein Synthesis - Cronodon
Despite the described attempts to get "something useful" out of Qb replicase, it appears likely that the main fascination with this enzyme will remain within the realm of basic science. The most intriguing questions may concern the precise mechanics of the interplay of the macromolecular components that make the system behave in the way it does. No doubt, knowledge of the three-dimensional structures of these components would be a great step forward toward this understanding. Efforts toward determining a structure of replicase by X-ray crystallography have not been successful thus far, but should be continued and extended to substructures like the core enzyme, S1 protein, and host factor. As the crystallography of RNA is still in its infancy, large RNA structures like the Qb plus and minus strand may not be accessible very soon, but interesting information could come from specific and well-studied small RNAs like MDV-1, especially if co-crystals with replicase and its subassemblies could be analyzed. In the meantime, the recent rapid advances in computer modeling of RNA structures (38) will continue and, together with phylogenetic comparisons and chemical or biochemical probing techniques, should give us improved models of template RNAs and their complexes with replicase. Similarly, we can expect that functional studies with site-directed RNA mutants, and especially the elegant evolutionary approach in which deficient mutants are allowed to evolve back to high viability (8), will continue to advance our understanding of this exemplary RNA-protein recognition process.
Toyobo Life Science Department - Modifing Enzyme ReverTra Ace-
Besides using the 3′ UTR for minus-sense RNA synthesis, the BaMV RdRp can also recognize 3′ terminal 77 nucleotides of the minus-strand for plus-sense RNA synthesis.
Custom PCR Array - SABiosciences
The interactions of replicase with the Qb minus strand and the replicating 6 S RNAs are expected to be different from those with the plus strand, because the former RNAs are efficient templates for core replicase in the absence of proteins S1 and Hfq. Deletion analysis of the Qb minus strand identified two structural features required for template activity (7). One consists of a sequence that folds into an imperfectly base-paired stem-loop structure located near the 5′-terminus of the minus strand. A highly homologous sequence is found in MDV-1 RNA (a 6 S RNA) and was characterized as an element essential for recognition of this template (28). The other essential element is formed by two short complementary sequences forming a helical stem by long-range base-pairing near the 3′-end of the minus strand. On the basis of stability calculations, the 3′-terminal H CCC(A) sequence, which represents another essential structure, appears in the minus strand to exist in an unpaired conformation, in agreement with the fact that this template does not require a host factor.
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