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.

RNase D is a 3'-exoribonuclease whose in vitro specificity has suggested that it is involved in the processing of tRNA precursors. Its in vivo role has remained unclear, however, because mutant cells devoid of the enzyme display no defect in growth or tRNA processing. To learn more about the structure and function of RNase D, we cloned the Escherichia coli rnd gene, which is thought to code for this enzyme. The rnd gene was isolated from a cosmid library based on elevated RNase D activity and was subcloned as a 1.4-kilobase-pair fragment in pUC18. Maxicell analysis of the cloned fragment revealed that a single protein of approximately 40 kilodaltons, which is the size of RNase D, was synthesized. The rnd gene is present as a single copy on the E. coli chromosome and is totally absent in a deletion mutant. Cells that harbored the cloned rnd gene displayed RNase D activity that was elevated as much as 20-fold over that of the wild type. As growth of the culture progressed, however, RNase D specific activity declined dramatically, together with a similar decrease in plasmid copy number. In contrast, no decrease in copy number was observed with an inactive rnd gene. Placement of the rnd gene downstream from the lac promoter led to inducible RNase D overexpression and concomitantly slowed cell growth. These findings support the idea that rnd is the structural gene for RNase D and indicate that elevated RNase D activity is deleterious to E. coli.

Cloning, characterization, and effects of overexpression of the Escherichia coli rnd gene encoding RNase D.

Transfer RNA nucleotidyltransferase repairs all transfer RNAs randomly

We have altered by site-directed mutagenesis the 3' terminal adenosine residue of a tRNA(Tyrsu3+) gene encoded on a single-copy plasmid and examined the consequences of these substitutions on suppressor activity in vivo. Our data show that mutant su3 genes containing 3'-CCC, -CCG, or -CCU termini instead of -CCA can be efficiently transcribed and processed in Escherichia coli to generate functional suppressor tRNAs. However, in contrast to normal tRNA genes, both tRNA nucleotidyltransferase and exoribonuclease activities are required to obtain suppression by the mutant tRNAs, indicating that removal of the incorrect 3' terminal residue and resynthesis of the normal -CCA terminus are occurring in this situation. In addition, a low level of suppressor activity and tRNA repair was found in cells devoid of tRNA nucleotidyltransferase, suggesting that an additional activity able to partially repair the 3' end of tRNA is present in E. coli. The use of mutant strains lacking one or several exoribonucleases revealed that the various RNAses have very different specificities for removal of incorrect 3' residues and that these differ greatly from their action on CCA-ending tRNA. These data show that the 3' terminal adenosine residue is necessary for tRNA function in vivo and that cells can compensate for its alteration by changes in the normal pathway of tRNA metabolism.

Substitution of the 3' terminal adenosine residue of transfer RNA in vivo

A relevant (rl) of the temperature-sensitive leucyl-tRNA synthetase mutant (tsHl) of Chinese hamster ovary cells has been analyzed. The enzyme in the revertant is also temperature sensitive, but its activity at every temperature tested is slightly higher than that of the mutant. Both tsHl and the revertant are sensitive to a decreased concentration of leucine in the growth medium, but the revertant is less sensitive than the mutant. The tsHl synthetase has a Km for leucine which is 80-fold higher at 39°C than that of wildtype CHO cells, whereas the Km of the rl enzyme is 20-fold higher than the wild type at this temperature.

Physiological and biochemical properties of a temperature-sensitive leucyl-tRNA synthetase mutant (tsHl) and revertant from Chinese hamster cells

Analysis of the upstream region of the Escherichia coli rnd gene encoding RNase D. Evidence for translational regulation of a putative tRNA processing enzyme.

Murray P. Deutscher

Papers

Cloning, characterization, and effects of overexpression of the Escherichia coli rnd gene encoding RNase D.

Transfer RNA nucleotidyltransferase repairs all transfer RNAs randomly

Substitution of the 3' terminal adenosine residue of transfer RNA in vivo

Physiological and biochemical properties of a temperature-sensitive leucyl-tRNA synthetase mutant (tsHl) and revertant from Chinese hamster cells

Analysis of the upstream region of the Escherichia coli rnd gene encoding RNase D. Evidence for translational regulation of a putative tRNA processing enzyme.