High-throughput mRNA sequencing (RNA-Seq) promises simultaneous transcript discovery and abundance estimation. However, this would require algorithms that are not restricted by prior gene annotations and that account for alternative transcription and splicing. Here we introduce such algorithms in an open-source software program called Cufflinks. To test Cufflinks, we sequenced and analyzed >430 million paired 75-bp RNA-Seq reads from a mouse myoblast cell line over a differentiation time series. We detected 13,692 known transcripts and 3,724 previously unannotated ones, 62% of which are supported by independent expression data or by homologous genes in other species. Over the time series, 330 genes showed complete switches in the dominant transcription start site (TSS) or splice isoform, and we observed more subtle shifts in 1,304 other genes. These results suggest that Cufflinks can illuminate the substantial regulatory flexibility and complexity in even this well-studied model of muscle development and that it can improve transcriptome-based genome annotation.

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Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation

Inefficient vascular supply and the resultant reduction in tissue oxygen tension often lead to neovascularization in order to satisfy the needs of the tissue. Examples include the compensatory development of collateral blood vessels in ischaemic tissues that are otherwise quiescent for angiogenesis and angiogenesis associated with the healing of hypoxic wounds. But the presumptive hypoxia-induced angiogenic factors that mediate this feedback response have not been identified. Here we show that vascular endothelial growth factor (VEGF; also known as vascular permeability factor) probably functions as a hypoxia-inducible angiogenic factor. VEGF messenger RNA levels are dramatically increased within a few hours of exposing different cell cultures to hypoxia and return to background when normal oxygen supply is resumed. In situ analysis of tumour specimens undergoing neovascularization show that the production of VEGF is specifically induced in a subset of glioblastoma cells distinguished by their immediate proximity to necrotic foci (presumably hypoxic regions) and the clustering of capillaries alongside VEGF-producing cells.

Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis.

The homeostasis of animals is regulated not only by the growth and differentiation of cells, but also by cell death through a process known as apoptosis. Apoptosis is mediated by members of the caspase family of proteases, and eventually causes the degradation of chromosomal DNA. A caspase-activated deoxyribonuclease (CAD) and its inhibitor (ICAD) have now been identified in the cytoplasmic fraction of mouse lymphoma cells. CAD is a protein of 343 amino acids which carries a nuclear-localization signal; ICAD exists in a long and a short form. Recombinant ICAD specifically inhibits CAD-induced degradation of nuclear DNA and its DNase activity. When CAD is expressed with ICAD in COS cells or in a cell-free system, CAD is produced as a complex with ICAD: treatment with caspase 3 releases the DNase activity which causes DNA fragmentation in nuclei. ICAD therefore seems to function as a chaperone for CAD during its synthesis, remaining complexed with CAD to inhibit its DNase activity; caspases activated by apoptotic stimuli then cleave ICAD, allowing CAD to enter the nucleus and degrade chromosomal DNA.

A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD

5-Noncoding sequences have been tabulated for 211 messenger RNAs from higher eukaryotic cells. The 5'-proximal AUG triplet serves as the initiator codon in 95% of the mRNAs examined. The most conspicuous conserved feature is the presence of a purine (most often A) three nucleotides upstream from the AUG initiator codon; only 6 of the mRNAs in the survey have a pyrimidine in that position. There is a predominance of C in positions -1, -2, -4 and -5, just upstream from the initiator codon. The sequence CCAGCCAUG (G) thus emerges as a consensus sequence for eukaryotic initiation sites. The extent to which the ribosome binding site in a given mRNA matches the -1 to -5 consensus sequence varies: more than half of the mRNAs in the tabulation have 3 or 4 nucleotides in common with the CCACC consensus, but only ten mRNAs conform perfectly.

Compilation and analysis of sequences upstream from the translational start site in eukaryotic mRNAs

DNA methylation is a major epigenetic modification of the genome that regulates crucial aspects of its function. Genomic methylation patterns in somatic differentiated cells are generally stable and heritable. However, in mammals there are at least two developmental periods-in germ cells and in preimplantation embryos-in which methylation patterns are reprogrammed genome wide, generating cells with a broad developmental potential. Epigenetic reprogramming in germ cells is critical for imprinting; reprogramming in early embryos also affects imprinting. Reprogramming is likely to have a crucial role in establishing nuclear totipotency in normal development and in cloned animals, and in the erasure of acquired epigenetic information. A role of reprogramming in stem cell differentiation is also envisaged. DNA methylation is one of the best-studied epigenetic modifications of DNA in all unicellular and multicellular organisms. In mammals and other vertebrates, methylation occurs predominantly at the symmetrical dinucleotide CpG (1-4). Symmetrical methylation and the discovery of a DNA methyltransferase that prefers a hemimethylated substrate, Dnmt1 (4), suggested a mechanism by which specific patterns of methylation in the genome could be maintained. Patterns imposed on the genome at defined developmental time points in precursor cells could be maintained by Dnmt1, and would lead to predetermined programs of gene expression during development in descendants of the precursor cells (5, 6). This provided a means to explain how patterns of differentiation could be maintained by populations of cells. In addition, specific demethylation events in differentiated tissues could then lead to further changes in gene expression as needed. Neat and convincing as this model is, it is still largely unsubstantiated. While effects of methylation on expression of specific genes, particularly imprinted ones (7) and some retrotransposons (8), have been demonstrated in vivo, it is still unclear whether or not methylation is involved in the control of gene expression during normal development (9-13). Although enzymes have been identified that can methylate DNA de novo (Dnmt3a and Dnmt3b) (14), it is unknown how specific patterns of methylation are established in the genome. Mechanisms for active demethylation have been suggested, but no enzymes have been identified that carry out this function in vivo (15-17). Genomewide alterations in methylation-brought about, for example, by knockouts of the methylase genes-result in embryo lethality or developmental defects, but the basis for abnormal development still remains to be discovered (7, 14). What is clear, however, is that in mammals there are developmental periods of genomewide reprogramming of methylation patterns in vivo. Typically, a substantial part of the genome is demethylated, and after some time remethylated, in a cell- or tissue-specific pattern. The developmental dynamics of these reprogramming events, as well as some of the enzymatic mechanisms involved and the biological purposes, are beginning to be understood. Here we look at what is known about reprogramming in mammals and discuss how it might relate to developmental potency and imprinting.

Epigenetic Reprogramming in Mammalian Development

THE muscular dystrophies are a group of hereditary disorders manifested by a progressive wasting of the skeletal muscles. In spite of extensive studies, the nature of the primary lesion is unknown (for review see ref. 1). Because of the complex interaction between tissues, it is difficult to study this question in vivo. Therefore attempts have been made to investigate this question in cultures of dystrophic muscles of human or animal origin. Tissue explants as well as monolayer primary cell cultures contain, in addition to the myogenic cells, a heterogeneous cell population, the composition of which might differ in normal and dystrophic muscle cultures. It is difficult in such experiments to distinguish between properties intrinsic to the myogenic cells and effects exerted by other cell types. Indeed, previous experiments have yielded conflicting conclusions2–6. We therefore tested the possibility of obtaining cell cultures consisting of pure populations of myogenic cells obtained from dystrophic muscles. The present report describes the isolation of a cloned population of such cells, derived from adult dystrophic mouse muscle, that can proliferate and differentiate in cell culture.

Serial passaging and differentiation of myogenic cells isolated from dystrophic mouse muscle

The nucleotide sequence of the rat beta-actin gene was determined. The gene codes for a protein identical to the bovine beta-actin. It has a large intron in the 5' untranslated region 6 nucleotides upstream from the initiator ATG, and 4 introns in the coding region at codons specifying amino acids 41/42, 121/122, 267, and 327/328. Unlike the skeletal muscle actin gene and many other actin genes, the beta-actin gene lacks the codon for Cys between the initiator ATG and the codon for the N-terminal amino acid of the mature protein. The usage of synonymous codons in the beta-actin gene is nonrandom, and is similar to that in the rat skeletal muscle and other vertebrate actin genes, but differs from the codon usage in yeast and soybean actin genes.

The nucleotide sequence of the rat cytoplasmic β–actin gene

https://www.pnas.org/content/pnas/61/2/477.full.pdf

David Yaffe

Papers

Serial passaging and differentiation of myogenic cells isolated from dystrophic mouse muscle

The nucleotide sequence of the rat cytoplasmic β–actin gene

Retention of differentiation potentialities during prolonged cultivation of myogenic cells

Alterations of enzymatic activities during muscle differentiation in vitro.

The in vitro cultivation and differentiation capacities of myogenic cell lines.