Irene Majerfeld

Affinity selection-amplification from randomized ribooligonucleotide pools.

Publisher Summary Selection-amplification introduced a new capability to the study of RNA and DNA. One could ask if any nucleic acid (less than a certain size) existed that could perform a particular biochemical function and recover that molecule for further study, along with its closely related functional relatives. Such exhaustive investigation of nucleic acid capabilities was unprecedented. This chapter discusses some of the methods and considerations required to carry out the purification, potentially ≈10 14 -fold, of a new RNA from a randomized pool of initial sequences. RNAs are fractionated (selected by affinity chromatography in this chapter) and the selected fraction is converted to complementary DNA (cDNA) that is amplified by polymerase chain reaction (PCR). Finally the DNA from the PCR is transcribed and the cycle is repeated. After the desired activity is observed in the pool or when selection has apparently succeeded, RNAs are cloned and sequenced. Individual clones are then characterized by appropriate structural and functional biochemical assays.

Identification of the regulatory subunit of a cAMP-dependent protein kinase in Dictyosteliumdiscoideum

Abstract The occurrence of a cytosolic cAMP-binding protein of an approximate molecular weight of 41,000 daltons was monitored in vegetative and developing amoebae of Dictyostelium discoideum by the use of the photoaffinity probe (32P) 8N3-cAMP. There was a large apparent increase in the amount of this binding protein during development; its molecular weight remained constant, if appropriate methods were employed for the disruption of the amoebae. Comigration during electrophoresis on two-dimensional gels identifies this cAMP-binding protein, photoaffinity-labeled in crude extracts, as the regulatory subunit of the cAMP-dependent protein kinase of D. discoideum .

Regulation of the synthesis of adenylate cyclase in Escherichia coli by the cAMP -- cAMP receptor protein complex.

SummaryThe synthesis of the adenylate cyclase [ATP pyrophosphatelyase-(cyclizing), E.C. 4.6.1.1.] of Escherichia coli, appears to be regulated negatively by the cAMP receptor protein CRP. This conclusion is based on a comparison of adenylate cyclase activities measured in vitro with the rates of cAMP synthesis by intact bacteria. The activity of adenylate cyclase, depending on conditions of growth, is also regulated by CRP; this effect, however, is indirect insofar as it is mediated by a protein or proteins under CRP control.

Nucleotides that are essential but not conserved; a sufficient L-tryptophan site in RNA

Conservation is often used to define essential sequences within RNA sites. However, conservation finds only invariant sequence elements that are necessary for function, rather than finding a set of sequence elements sufficient for function. Biochemical studies in several systems-including the hammerhead ribozyme and the purine riboswitch-find additional elements, such as loop-loop interactions, required for function yet not phylogenetically conserved. Here we define a critical test of sufficiency: We embed a minimal, apparently sufficient motif for binding the amino acid tryptophan in a random-sequence background and ask whether we obtain functional molecules. After a negative result, we use a combination of three-dimensional structural modeling, selection, designed mutations, high-throughput sequencing, and bioinformatics to explore functional insufficiency. This reveals an essential unpaired G in a diverse structural context, varied sequence, and flexible distance from the invariant internal loop binding site identified previously. Addition of the new element yields a sufficient binding site by the insertion criterion, binding tryptophan in 22 out of 23 tries. Random insertion testing for site sufficiency seems likely to be broadly revealing.

An increase of cAMP-dependent protein kinase during development in Polysphondylium pallidum☆

Polysphondylium pallidum is a cellular slime mold in which, unlike in Dictyostelium discoideum, cAMP is not the chemotactic agent. The occurrence of a cAMP-dependent protein kinase in D. discoideum was demonstrated earlier and we suggested that it may mediate the intracellular effects of cAMP on the development of the organism, particularly since an increase in the amount of the enzyme during development was noted. In D. discoideum cAMP plays a dual role insofar as it serves both as chemotactic agent and as second messenger; it was of interest therefore, to determine whether a cAMP-dependent protein kinase occurred in P. pallidum. We found a cAMP-dependent protein kinase in P. pallidum using Kemptide as substrate. The regulatory subunit of the enzyme has an apparent molecular weight of 41,000 and seems to be similar in its properties with that isolated earlier from D. discoideum. The cAMP-dependent protein kinase catalytic subunits from the two species are also similar. Furthermore, there is a developmentally regulated, parallel, two- to threefold increase in the two subunits of the cAMP-dependent protein kinase in P. pallidum. The increase occurs before aggregates are formed. These findings are compatible with a role of the intracellular cAMP and of the cAMP-dependent protein kinase in the development of P. pallidum.