Tens of millions of short peptides can be easily surveyed for tight binding to an antibody, receptor or other binding protein using an "epitope library." The library is a vast mixture of filamentous phage clones, each displaying one peptide sequence on the virion surface. The survey is accomplished by using the binding protein to affinity-purify phage that display tight-binding peptides and propagating the purified phage in Escherichia coli. The amino acid sequences of the peptides displayed on the phage are then determined by sequencing the corresponding coding region in the viral DNA's. Potential applications of the epitope library include investigation of the specificity of antibodies and discovery of mimetic drug candidates.

https://science.sciencemag.org/content/sci/249/4967/386.full.pdf

Searching for Peptide Ligands With an Epitope Library

Epitope tagging of proteins as a strategy for the analysis of function, interactions and the subcellular distribution of proteins has become widely used. In the yeast Saccharomyces cerevisiae, molecular biological techniques have been developed that use a simple PCR-based strategy to introduce epitope tags to chromosomal loci (Wach et al., 1994). To further employ the power of this strategy, a variety of novel tags was constructed. These tags were combined with different selectable marker genes, resulting in PCR amplificable modules. Only one set of primers is required for the amplification of any module. Furthermore, convenient laboratory techniques are described that facilitate the genetic manipulations of yeast strains, as well as the analysis of the epitope-tagged proteins.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/%28SICI%291097-0061%28199907%2915%3A10B%3C963%3A%3AAID-YEA399%3E3.0.CO%3B2-W

Epitope tagging of yeast genes using a PCR‐based strategy: more tags and improved practical routines

These revised recommendations regarding vaccinia (smallpox) vaccine update the previous Advisory Committee on Immunization Practices (ACIP) recommendations (MMWR 1991;40; No. RR-14:1-10) and include current information regarding the nonemergency use of vaccinia vaccine among laboratory and health-care workers occupationally exposed to vaccinia virus, recombinant vaccinia viruses, and other Orthopoxviruses that can infect humans. In addition, this report contains ACIP's recommendations for the use of vaccinia vaccine if smallpox (variola) virus were used as an agent of biological terrorism or if a smallpox outbreak were to occur for another unforeseen reason.

Vaccinia (smallpox) vaccine: recommendations of the Advisory Committee on Immunization Practices (ACIP), 2001.

Rapid degradation of c-fos proto-oncogene mRNA is crucial for transient c-fos gene expression. Experiments were performed to investigate the cellular mechanisms responsible for the extremely short half-life of human c-fos mRNA in growth-factor-stimulated fibroblasts. These experiments demonstrate the existence of two distinct cellular pathways for rapid c-fos mRNA degradation. Each of these pathways recognizes a different, functionally independent instability determinant within the c-fos transcript. One instability determinant, which is located within the c-fos 3'-untranslated region, is a 75-nucleotide AU-rich segment. Insertion of this element into beta-globin mRNA markedly reduces the half-life of that normally long-lived message. Nevertheless, specific deletion of the AU-rich element from c-fos mRNA has little effect on the transcript's cytoplasmic half-life due to the presence of the other c-fos instability determinant, which is located in the protein-coding segment of the c-fos message. Examination of mRNA decay in cells treated with transcription inhibitors indicates that one c-fos mRNA degradation pathway is dependent on RNA synthesis, whereas the other is not.

The c-fos transcript is targeted for rapid decay by two distinct mRNA degradation pathways.

The mechanisms by which c-fos mRNA is targeted for decay have been examined. Rapid removal of the poly(A) tail occurs before the transcribed portion of the c-fos message is degraded. Identification of the determinants that mediate c-fos message deadenylation reveals that they coincide directly with previously characterized determinants of c-fos mRNA instability, one in the protein-coding region and the other an AU-rich element (ARE) in the 3'-untranslated region. Insertion of either of these c-fos instability elements into the stable beta-globin message confers the property of rapid deadenylation. Mutation of the ARE indicates that this sequence controls two steps in the process of c-fos mRNA degradation: removal of the poly(A) tail, which does not require intact AUUUA pentanucleotides within the ARE, and subsequent degradation of the transcribed portion of the message, which appears to be dependent on the AUUUA pentanucleotides. These results indicate that structurally distinct instability determinants within the transcribed portion of labile messages can function by promoting rapid removal of the poly(A) tail as a first step in the decay process.

/pdf/two-distinct-destabilizing-elements-in-the-c-fos-message-2syh5sstwn.pdf

Two distinct destabilizing elements in the c-fos message trigger deadenylation as a first step in rapid mRNA decay.

Rabies is one of the oldest diseases know to man, but its successful control has remained elusive. Although effective vaccines of tissue culture origin against rabies do exist, such preparations are expensive. Live vaccinia virus (VV) recombinants expressing influenza or hepatitis B antigens have recently been used to immunize against these diseases. We have now used this approach to produce a novel rabies vaccine. We first altered the rabies glycoprotein cDNA by site-directed mutagenesis and removed the poly(dG) tail. We then aligned the modified cDNA with an early VV promoter sequence inserted within a cloned copy of the vaccinia thymidine kinase gene and transfected this plasmid into VV-infected cells. Recombination between the virus and the plasmid resulted in a recombinant virus harbouring the rabies glycoprotein cDNA. Inoculation of rabbits with the live recombinant virus induced high titres of rabies virus-neutralizing antibodies, and scarification with the recombinant VV protected mice against challenge with street rabies virus.

Expression of rabies virus glycoprotein from a recombinant vaccinia virus

Current procedures for inserting linker oligonucleotides between DNA termini involve terminal addition of linker duplexes followed by restriction enzyme cleavage and religation. Such methods have the disadvantage that target DNA molecules containing internal recognition sites are cut during the linker cleavage step. We describe a method for adding linker oligonucleotides to DNA termini which eliminates the requirement for subsequent restriction enzyme treatment. Nonphosphorylated linker duplexes are ligated to target DNA termini on one strand alone. Removal of the unligated strand generates a single-stranded protrusion at each terminus. Two such tailed termini may now be linked by annealing their single-stranded complementary tails, resulting in a hybrid DNA molecule containing a single linker insertion. Each step of the procedure has been studied to optimize the recovery of recombinant molecules, and we present examples of how the method may be applied.

S. Skory

Papers

Expression of rabies virus glycoprotein from a recombinant vaccinia virus

Linker tailing: unphosphorylated linker oligonucleotides for joining DNA termini