A new method for determining nucleotide sequences in DNA is described. It is similar to the “plus and minus” method [Sanger, F. & Coulson, A. R. (1975) J. Mol. Biol. 94, 441-448] but makes use of the 2′,3′-dideoxy and arabinonucleoside analogues of the normal deoxynucleoside triphosphates, which act as specific chain-terminating inhibitors of DNA polymerase. The technique has been applied to the DNA of bacteriophage ϕX174 and is more rapid and more accurate than either the plus or the minus method.

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DNA sequencing with chain-terminating inhibitors

Oligodendrocytes, the myelin-forming cells of the central nervous system (CNS), and astrocytes constitute macroglia. This review deals with the recent progress related to the origin and differentiation of the oligodendrocytes, their relationships to other neural cells, and functional neuroglial interactions under physiological conditions and in demyelinating diseases. One of the problems in studies of the CNS is to find components, i.e., markers, for the identification of the different cells, in intact tissues or cultures. In recent years, specific biochemical, immunological, and molecular markers have been identified. Many components specific to differentiating oligodendrocytes and to myelin are now available to aid their study. Transgenic mice and spontaneous mutants have led to a better understanding of the targets of specific dys- or demyelinating diseases. The best examples are the studies concerning the effects of the mutations affecting the most abundant protein in the central nervous myelin, the p...

/pdf/biology-of-oligodendrocyte-and-myelin-in-the-mammalian-3ggbjcby4w.pdf

Biology of Oligodendrocyte and Myelin in the Mammalian Central Nervous System

Improvements in genomic and molecular methods are expanding the range of potential applications for circulating tumour DNA (ctDNA), both in a research setting and as a 'liquid biopsy' for cancer management. Proof-of-principle studies have demonstrated the translational potential of ctDNA for prognostication, molecular profiling and monitoring. The field is now in an exciting transitional period in which ctDNA analysis is beginning to be applied clinically, although there is still much to learn about the biology of cell-free DNA. This is an opportune time to appraise potential approaches to ctDNA analysis, and to consider their applications in personalized oncology and in cancer research.

/pdf/liquid-biopsies-come-of-age-towards-implementation-of-3ovu7jzn3f.pdf

Liquid biopsies come of age: towards implementation of circulating tumour DNA

Thousands of loci in the human and mouse genomes give rise to circular RNA transcripts; at many of these loci, the predominant RNA isoform is a circle. Using an improved computational approach for circular RNA identification, we found widespread circular RNA expression in Drosophila melanogaster and estimate that in humans, circular RNA may account for 1% as many molecules as poly(A) RNA. Analysis of data from the ENCODE consortium revealed that the repertoire of genes expressing circular RNA, the ratio of circular to linear transcripts for each gene, and even the pattern of splice isoforms of circular RNAs from each gene were cell-type specific. These results suggest that biogenesis of circular RNA is an integral, conserved, and regulated feature of the gene expression program.

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Cell-type specific features of circular RNA expression.

Post-transcriptional gene regulation (PTGR) concerns processes involved in the maturation, transport, stability and translation of coding and non-coding RNAs. RNA-binding proteins (RBPs) and ribonucleoproteins coordinate RNA processing and PTGR. The introduction of large-scale quantitative methods, such as next-generation sequencing and modern protein mass spectrometry, has renewed interest in the investigation of PTGR and the protein factors involved at a systems-biology level. Here, we present a census of 1,542 manually curated RBPs that we have analysed for their interactions with different classes of RNA, their evolutionary conservation, their abundance and their tissue-specific expression. Our analysis is a critical step towards the comprehensive characterization of proteins involved in human RNA metabolism.

/pdf/a-census-of-human-rna-binding-proteins-1mii7pfs5y.pdf

A census of human RNA-binding proteins.

RNases play a central role in the metabolism of RNA. Among the many reactions catalyzed by these enzymes are those that separate individual entities of multicomponent transcripts, those that generate the mature 5' and 3' termini of RNAs, and those that remove introns. RNases also degrade RNA molecules that are no longer needed by the cell or that are damaged in some way, and under some conditions they also digest RNAs to supply nucleotides for continued RNA synthesis. As knowledge of the details of RNA metabolism has expanded, there has been a growing realization that many RNases with distinct specificities might be needed to catalyze the diverse reactions undergone by RNA molecules. Our understanding of this problem has progressed most rapidly in studies with Escherchia coli, so that for this organism close to 20 RNases are already known (17), and others undoubtedly exist. Exoribonucleases are a class of RNases that digest RNA molecules from their ends. The exoribonucleases currently known generate mononucleotide residues which can be reutilized for RNA synthesis. Exoribonucleases have been implicated in a number of RNA metabolic events, including RNA maturation and RNA degradation. In the case of RNA maturation, exoribonucleases participate in the final processing steps, trimming terminal residues from certain RNA precursors. Exoribonucleases also play a role in degrading unwanted RNA molecules to the mononucleotide level. In E. coli, two exoribonucleases that act nonspecifically in vitro on all types of RNA molecules, PNPase and RNase II, have been known for many years (45, 66). The properties of these two enzymes, and those of nonspecific exoribonucleases identified from other organisms, incorrectly colored the early thinking about this class of enzymes. However, in contrast to the small number of nonspecific, degradative exoribonucleases initially identified, it is now clear that a single cell can contain a large number of distinct proteins that function to digest RNA exonucleolytically. This minireview will discuss the information that has come to light in recent years concerning this family of enzymes from E. coli.

Promiscuous exoribonucleases of Escherichia coli

Escherichia coli contains multiple exoribonucleases. Strains lacking the exoribonucleases RNase II, D, BN, T, and PH are inviable. The introduction of a chromosomal, wild-type copy of the gene for any one of these enzymes is sufficient to allow cell growth, with the enzymes being in the following order of effectiveness: RNase T > RNase PH > RNase D > RNase II > RNase BN. The data indicate that these five exoribonucleases functionally overlap in vivo and that any one of them can take over the functions of all the others, although with various efficiencies.

The presence of only one of five exoribonucleases is sufficient to support the growth of Escherichia coli.

Our knowledge of the 3' processing of tRNA precursors is severely limited. Although six exoribonucleases able to act on Escherichia coli tRNA precursors in vitro have been identified, their involvement in tRNA maturation in vivo has not been demonstrated. Here we show, using a wide range of multiple RNase-deficient strains and a quantitative suppression assay, that at least five of these enzymes--RNase II, RNase D, RNase BN, RNase T, and RNase PH--can participate in the synthesis of functional tRNA(Tyr)su+3 in vivo. Moreover, any one of the five RNases is sufficient to allow tRNA processing to proceed although with varying effectiveness. Examination of the level of aminoacylation of tRNA isolated from RNase-deficient strains suggested that tRNA precursors accumulate in the most defective cells. These data indicate that exoribonucleases are required for tRNA maturation in vivo and that there is a high degree of functional overlap among the enzymes. These studies contribute to the identification of all the ...

Murray P. Deutscher

Papers

Promiscuous exoribonucleases of Escherichia coli

The presence of only one of five exoribonucleases is sufficient to support the growth of Escherichia coli.

Multiple exoribonucleases are required for the 3' processing of Escherichia coli tRNA precursors in vivo.

E. coli RNases: making sense of alphabet soup.

Multiple exoribonucleases catalyze maturation of the 3' terminus of 16S ribosomal RNA (rRNA).