http://biology.hunter.cuny.edu/molecularbio/Class%20Materials%20Spring%202012%20Biol302/Lecture%203/Kozak%20Test%20of%20rules.pdf

Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes.

The reverse transcription polymerase chain reaction (RT-PCR) is the most sensitive method for the detection of low-abundance mRNA, often obtained from limited tissue samples. However, it is a complex technique, there are substantial problems associated with its true sensitivity, reproducibility and specificity and, as a quantitative method, it suffers from the problems inherent in PCR. The recent introduction of fluorescence-based kinetic RT-PCR procedures significantly simplifies the process of producing reproducible quantification of mRNAs and promises to overcome these limitations. Nevertheless, their successful application depends on a clear understanding of the practical problems, and careful experimental design, application and validation remain essential for accurate quantitative measurements of transcription. This review discusses the technical aspects involved, contrasts conventional and kinetic RT-PCR methods for quantitating gene expression and compares the different kinetic RT-PCR systems. It illustrates the usefulness of these assays by demonstrating the significantly different levels of transcription between individuals of the housekeeping gene family, glyceraldehyde-3-phosphate-dehydrogenase (GAPDH).

/pdf/absolute-quantification-of-mrna-using-real-time-reverse-2kyvkvy0jn.pdf

Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays.

/pdf/purification-and-characterization-2y44lhntkf.pdf

Purification and Characterization

Tremendous progress has been made in understanding the molecular basis of the antiviral actions of interferons (IFNs), as well as strategies evolved by viruses to antagonize the actions of IFNs. Furthermore, advances made while elucidating the IFN system have contributed significantly to our understanding in multiple areas of virology and molecular cell biology, ranging from pathways of signal transduction to the biochemical mechanisms of transcriptional and translational control to the molecular basis of viral pathogenesis. IFNs are approved therapeutics and have moved from the basic research laboratory to the clinic. Among the IFN-induced proteins important in the antiviral actions of IFNs are the RNA-dependent protein kinase (PKR), the 2',5'-oligoadenylate synthetase (OAS) and RNase L, and the Mx protein GTPases. Double-stranded RNA plays a central role in modulating protein phosphorylation and RNA degradation catalyzed by the IFN-inducible PKR kinase and the 2'-5'-oligoadenylate-dependent RNase L, respectively, and also in RNA editing by the IFN-inducible RNA-specific adenosine deaminase (ADAR1). IFN also induces a form of inducible nitric oxide synthase (iNOS2) and the major histocompatibility complex class I and II proteins, all of which play important roles in immune response to infections. Several additional genes whose expression profiles are altered in response to IFN treatment and virus infection have been identified by microarray analyses. The availability of cDNA and genomic clones for many of the components of the IFN system, including IFN-alpha, IFN-beta, and IFN-gamma, their receptors, Jak and Stat and IRF signal transduction components, and proteins such as PKR, 2',5'-OAS, Mx, and ADAR, whose expression is regulated by IFNs, has permitted the generation of mutant proteins, cells that overexpress different forms of the proteins, and animals in which their expression has been disrupted by targeted gene disruption. The use of these IFN system reagents, both in cell culture and in whole animals, continues to provide important contributions to our understanding of the virus-host interaction and cellular antiviral response.

Antiviral Actions of Interferons

The N-methyl d-aspartate (NMDA) receptor subtype of glutamate-gated ion channels possesses high calcium permeability and unique voltage-dependent sensitivity to magnesium and is modulated by glycine Molecular cloning identified three complementary DNA species of rat brain, encoding NMDA receptor subunits NMDAR2A (NR2A), NR2B, and NR2C, which are 55 to 70% ientical in sequence These are structurally related, with less than 20% sequence identity, to other excitatory amino acid receptor subunits, including the NMDA receptor subunit NMDAR1 (NR1) Upon expression in cultured cells, the new subunits yielded prominent, typical glutamate-and NMDA-activated currents only when they were in heteromeric configurations with NR1 NR1-NR2A and NR1-NR2C channels differed in gating behavior and magnesium sensitivity Such heteromeric NMDA receptor subtypes may exist in neurons, since NR1 messenger RNA is synthesized throughout the mature rat brain, while NR2 messenger RNA show a differential distribution

https://science.sciencemag.org/content/sci/256/5060/1217.full.pdf

Heteromeric NMDA receptors: Molecular and functional distinction of subtypes

A simple and efficient method for synthesizing pure single stranded RNAs of virtually any structure is described. This in vitro transcription system is based on the unusually specific RNA synthesis by bacteriophage SP6 RNA polymerase which initiates transcription exclusively at an SP6 promoter. We have constructed convenient cloning vectors that contain an SP6 promoter immediately upstream from a polylinker sequence. Using these SP6 vectors, optimal conditions have been established for in vitro RNA synthesis. The advantages and uses of SP6 derived RNAs as probes for nucleic acid blot and solution hybridizations are demonstrated. We show that single stranded RNA probes of a high specific activity are easy to prepare and can significantly increase the sensitivity of nucleic acid hybridization methods. Furthermore, the SP6 transcription system can be used to prepare RNA substrates for studies on RNA processing (1,5,9) and translation (see accompanying paper).

Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter

Identification and Cloning of Localized Maternal RNAs From Xenopus Eggs

https://www.cell.com/cell/pdf/0092-8674(87)90238-8.pdf

Antisense RNA injections in fertilized frog eggs reveal an RNA duplex unwinding activity

Localized maternal mRNAs in Xenopus laevis eggs.

Single-stranded RNA molecules of defined sequence are useful as substrates for the investigation of such cellular processes as RNA processing and translation, and when highly labeled they serve as extremely efficient hybridization probes. An in vitro transcription system based on the unusual properties of SP6 RNA polymerase facilitates the synthesis of homogeneous single-stranded RNA molecules of any desired sequence. In this chapter we describe the SP6 transcription reaction in detail and point out the advantages and disadvantages of the use of single-stranded RNA molecules as substrates and as hybridization probes. In addition, we review recent studies on the use of SP6 transcripts as anti-sense RNAs that can hybridize to mRNAs in vivo and thereby block translation of a particular gene product.

M.R. Rebagliati

Papers

Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter

Identification and Cloning of Localized Maternal RNAs From Xenopus Eggs

Antisense RNA injections in fertilized frog eggs reveal an RNA duplex unwinding activity

Localized maternal mRNAs in Xenopus laevis eggs.

Synthesis of Hybridization Probes and RNA Substrates with SP6 RNA Polymerase