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.

relA overcomes the slow growth of cca mutants

A novel mechanism of ribonuclease regulation: GcvB and Hfq stabilize the mRNA that encodes RNase BN/Z during exponential phase

RNase II is the most active exoribonuclease in Escherichia coli cell extracts. Yet, its removal appears to have no deleterious effect on growing cells. Here, we show that RNase II is required for cell survival during prolonged stationary phase and upon starvation. The absence of RNase II leads to greatly increased rRNA degradation and to the accumulation of rRNA fragments, both of which lead to a decline in cell survival. The deleterious effects of RNase II removal can be completely reversed by the simultaneous absence of a second exoribonuclease, RNase PH, an enzyme known to be required to initiate ribosome degradation in starving cells. We have now found that the role of RNase II in this process is to regulate the amount of RNase PH present in starving cells, and it does so at the level of RNase PH stability. RNase PH normally decreases as much as 90% during starvation because the protein is unstable under these conditions; however, in the absence of RNase II the amount of RNase PH remains relatively unchanged. Based on these observations, we propose that in the presence of RNase II, nutrient deprivation leads to a dramatic reduction in the amount of RNase PH, thereby limiting the extent of rRNA degradation and ensuring cell survival during this stress. In the absence of RNase II, RNase PH levels remain high, leading to excessive ribosome loss and ultimately to cell death. These findings provide another example of RNase regulation in response to environmental stress.

/pdf/rnase-ii-regulates-rnase-ph-and-is-essential-for-cell-2n7b9t0uoi.pdf

RNase II regulates RNase PH and is essential for cell survival during starvation and stationary phase.

A functional mutant of tRNAArg2 with ten extra nucleotides in its TFC arm

Publisher Summary This chapter describes the procedures for the purification of the enzyme from Escherichia coli , and rabbit liver. This procedure yields a preparation of the enzyme from rabbit liver that is homogeneous and about 30,000-fold purified. The assay for tRNA nucleotidyltransferase measures the incorporation of radioactive, acid-soluble adenosine triphosphate (ATP) or cytosine triphosphate (CTP) into an acid-insoluble product. Purified preparations of rabbit liver tRNA nucleotidyltransferase contain a poly(C) polymerase activity as an integral part of the protein. This activity can attach long sequences of cytosine monophosphate (CMP) residues to any intact or partially degraded tRNA molecule or to rRNA. The rate of this activity under the usual assay conditions is only about 1% of the normal CMP incorporation and is generally not detected.

Murray P. Deutscher

Papers

relA overcomes the slow growth of cca mutants

A novel mechanism of ribonuclease regulation: GcvB and Hfq stabilize the mRNA that encodes RNase BN/Z during exponential phase

RNase II regulates RNase PH and is essential for cell survival during starvation and stationary phase.

A functional mutant of tRNAArg2 with ten extra nucleotides in its TFC arm

[58] Rabbit liver tRNA nucleotidyltransferase