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.

Exoribonuclease and Endoribonuclease Activities of RNase BN/RNase Z both Function in Vivo

Essentially all (>97%) of the transfer ribonucleic acid (tRNA) in log-phase and sporulating cells of Bacillus megaterium contains a complete 3′-cytidyl-cytidyl-adenosine terminus. However, about one-third of the tRNA in the dormant spore lacks the 3′-terminal adenosine 5′-monophosphate (AMP) residue, and some of the adjacent cytosine monophosphate residues are also missing. Examination of specific tRNAs indicated that those specific for isoleucine, leucine, and methionine are missing 30 to 40% of their terminal residue, whereas tRNAs specific for tyrosine lack 88% of the 3′-terminal AMP. Defective spore tRNA is not degraded during germination, but the missing residues are added back in the first minutes of the process. The enzyme catalyzing the addition reaction, tRNA nucleotidyltransferase, is present in the dormant spore at a level similar to that found in the vegetative cell.

Absence of 3′-Terminal Residues from Transfer Ribonucleic Acid of Dormant Spores of Bacillus megaterium

The Helicase Activity of Ribonuclease R Is Essential for Efficient Nuclease Activity.

Proteins are fragile molecules that often require great care during purification to ensure that they remain intact and fully active. Nowadays, many proteins are also purified in small amounts under denaturing conditions by various gel electrophoretic techniques, such that inactive proteins are obtained. But even here, it is usually advantageous to maintain the protein in an intact form. In the case of enzymes, and other proteins with assayable biological activities, maintenance of activity is generally of prime importance, both for following the protein during purification and for subsequent studies of function. This chapter will focus on the major points to keep in mind with regard to maintaining the stability of a protein during purification and storage. Various other chapters describe in detail stabilization procedures for specific biological systems and specific classes of proteins.

Chapter 10 Maintaining Protein Stability

Publisher Summary All tRNA molecules contain, at the 3′ terminus, the identical-CCA sequence that is required for their acceptor and transfer functions. In prokaryotic cells, no discrete pattern has emerged. In laboratory strains of Escherichia coli , all tRNA genes sequenced encode the -CCA sequence, and mutant strains devoid of tRNA nucleotidyltransferase retain viability, although they grow slowly. The most convenient tRNA substrate for routine use during purification is commercial bakers' yeast tRNA. Generally, this material contains about 50% tRNA-CC and is active with tRNA nucleotidyltransferases from most sources. If more defined preparations of tRNA-CC, tRNA-C, or tRNA-N are required, they can be prepared by single or multiple cycles of periodate oxidation and alkaline phosphatase treatment of any tRNA. The filters are placed along the side of miniscintillation vials, dried under an infrared lamp for 10 minutes, and counted in a scintillation counter with 3 ml of scintillation fluid. Under certain conditions, tRNA nucleotidyltransferases can make errors in nucleotide incorporation, and these anomalous reactions have been used successfully for the synthesis of tRNA molecules with altered 3′ termini.

Murray P. Deutscher

Papers

Exoribonuclease and Endoribonuclease Activities of RNase BN/RNase Z both Function in Vivo

Absence of 3′-Terminal Residues from Transfer Ribonucleic Acid of Dormant Spores of Bacillus megaterium

The Helicase Activity of Ribonuclease R Is Essential for Efficient Nuclease Activity.

Chapter 10 Maintaining Protein Stability

Transfer RNA nucleotidyltransferase.