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.

/pdf/dna-sequencing-with-chain-terminating-inhibitors-t64rmetxr4.pdf

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.

/pdf/cell-type-specific-features-of-circular-rna-expression-4ao4gawmcm.pdf

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.

In earlier studies it was shown that the mammalian translation system is highly organized in vivo and that the intermediates in the process, aminoacyl-tRNAs, are channeled--i.e., they are directly transferred from the aminoacyl-tRNA synthetases to the elongation factor to the ribosomes without dissociating into the cellular fluid. Here, we examine whether spent tRNAs leaving the ribosome enter the fluid phase or are transferred directly to their cognate aminoacyl-tRNA synthetases to complete a channeled tRNA cycle. Using a permeabilized CHO cell system that closely mimics living cells, we find that there is no leakage of endogenous tRNA during many cycles of translation, and protein synthesis remains linear during this period, even though free aminoacyl-tRNA is known to rapidly equilibrate between the inside and outside of these cells. We also find that exogenous tRNA and periodate-oxidized tRNA have no effect on protein synthesis in this system, indicating that they do not enter the translation machinery, despite the fact that exogenous tRNA rapidly distributes throughout the cells. Furthermore, most of the cellular aminoacyl-tRNA synthetases function only with endogenous tRNAs, although a portion can use exogenous tRNA molecules. However, aminoacylation of these exogenous tRNAs is strongly inhibited by oxidized tRNA; this inhibitor has no effect on endogenous aminoacylation. On the basis of these and the earlier observations, we conclude that endogenous tRNA is never free of the protein synthetic machinery at any stage of the translation process and, consequently, that there is a channeled tRNA cycle during protein synthesis in mammalian cells.

A channeled tRNA cycle during mammalian protein synthesis

Enzymatic synthesis of deoxyribonucleic acid. XXXIV. Termination of chain growth by a 2',3'-dideoxyribonucleotide

In recent years there has been a dramatic shift in our thinking about ribonucleases (RNases). Although they were once considered to be nonspecific, degradative enzymes, it is now clear that RNases play a central role in every aspect of cellular RNA metabolism, including decay of mRNA, conversion of RNA precursors to their mature forms, and end-turnover of certain RNAs. Recognition of the importance of this class of enzymes has led to an explosion of work and the establishment of significant new concepts. Thus, we now realize that RNases, both endoribonucleases and exoribonucleases, can be highly specific for particular sequences or structures. It has also become apparent that a single cell can contain a large number of distinct RNases, approaching as many as 20 members, often with overlapping specificities. Some RNases also have been found to be components of supramolecular complexes and to function in concert with other enzymes to carry out their role in RNA metabolism. This review focuses on the exoribonucleases, both prokaryotic and eukaryotic, and details their structure, catalytic properties, and physiological function.

Exoribonucleases and their multiple roles in RNA metabolism.

Although evidence suggests that the protein synthetic machinery is organized within cells, this point has been difficult to prove because any organization that might exist is lost upon preparation of the cell-free systems usually used to study translation in vitro. To examine this process under conditions more representative of the intact cell, we have developed an active protein-synthesizing system using Chinese hamster ovary (CHO) cells permeabilized with the plant glycoside saponin. This procedure renders cells permeable to trypan blue and exogenous tRNA, but there is little release of endogenous macromolecules. Protein synthesis in this system proceeds at the same rate as that in intact cells and is about 40-fold faster than that in a cell-free system prepared from the same cells. Active protein synthesis in this system requires the addition of only Mg2+, K+, and creatine phosphate, with a small further stimulation by ATP and an amino acid mixture; no exogenous macromolecules are necessary. The proteins synthesized in this system are indistinguishable from those made by the intact cell, and the channeling of aminoacyl-tRNA observed in vivo is maintained. Our data suggest that the permeabilized cell system retains the protein-synthesizing capabilities of the intact cell and presumably its internal structure as well. Studies with this system demonstrate that the protein-synthesizing apparatus is highly organized and that its macromolecular components are not freely diffusible in mammalian cells.

Supramolecular organization of the mammalian translation system

Publisher Summary This chapter describes 3’ maturation and repair, with particular emphasis on the ribonucleases and tRNA nucleotidyltransferase, the enzymes that participate in these processes. All tRNA molecules are synthesized initially as tRNA precursors containing additional residues at their 5’ and 3‘ termini that must be removed to generate active tRNAs. The strict requirement for an intact -C-C-A sequence for the proper functioning of tRNA necessitates specific and accurate processing machinery for 3’ maturation and also a mechanism to repair tRNA molecules with faulty 3’ termini. For precursors in which -C-C-A residues are already present (type-I precursors), 3’ maturation would only require removal of residues following the mature terminus, either by exonucleolytic trimming or by a specific endonucleolytic cut following the 3’ terminal adenosine. Such enzymes are known. In addition, there are differences in 3’-terminal structure between most prokaryotic and eukaryotic tRNA precursors that introduce an additional level of complexity to this process.

Murray P. Deutscher

Papers

A channeled tRNA cycle during mammalian protein synthesis

Enzymatic synthesis of deoxyribonucleic acid. XXXIV. Termination of chain growth by a 2',3'-dideoxyribonucleotide

Exoribonucleases and their multiple roles in RNA metabolism.

Supramolecular organization of the mammalian translation system

Ribonucleases, tRNA nucleotidyltransferase, and the 3' processing of tRNA.