Genes whose expression is growth factor regulated are likely to be important components in the mechanisms controlling cell proliferation and differentiation. With the aim of identifying some of those genes, a A cDNA library was prepared with poly(A)+ RNA from quiescent NIH 3T3 cells stimulated with serum for 4 h in the presence of cycloheximide. Differential screening of approximately 200,000 recombinant phage plaques revealed 2,540 clones that cross hybridized preferentially with [32P]cDNA derived from RNA of stimulated cells rather than with cDNA derived from nonstimulated cells. Cross hybridization of these clones identified 82 independent sequences, including c-fos and c-myc. Seventy-one clones were further studied. Analysis of the changes in transcription and mRNA levels after serum stimulation demonstrated that the kinetics and extent of the induction vary dramatically between the different genes. Cycloheximide in all cases superinduced the mRNA levels by two mechanisms, inhibiting the shutoff of transcription and prolonging the half-lives of the mRNAs. Our results showed that induction of proliferation is accompanied by the onset of a complex genetic program.A detailed knowledge of the biochemical events and the identification of the genes that are involved in the response to growth factors is currently being accumulated. A subset of these genes may play a critical role in cellular transformation. It is well established that the synthesis of new mRNA in quiescent cells is required for the cells to respond to mitogens and progress through Gl and enter the S phase (1). Furthermore, Gl seems to be a critical point of control of proliferation, where normal cells deprived of growth factors halt cycling, while transformed cells do not, suggesting that regulatory genes uncontrolled in the neoplastic phenotype are expressed during this transition. It has been estimated that in mouse fibroblasts, 3% of the mRNA species in logarithmically growing cells are absent in quiescent cells (49); thus, a set of these genes must be under the control of growth factors to allow cell proliferation. Evidence that gene expression could be controlled by the binding of a specific ligand to its receptor initially came from studies done with beta interferon (27,28) which demonstrated the transcriptional activation of two genes in human cells after interferon treatment. These findings were further supported by the interesting observation that the proto-oncogenes c-fos and c-myc present an immediate change in expression after stimulation of fibroblasts with growth factors (4,8,14,23,25,32), emphasizing also that important growth-controlling genes operate during the GO-to-Gl transition. Both genes are induced in the presence of protein synthesis inhibitors, demonstrating that their induction is a direct consequence of the growth factor-receptor interaction. Expression of c-fos is undetectable during the cell cycle (3), suggesting that its expression could be essential during the transition from GO to Gl and not for continuously growing ...

A Modular Open‐Source Assembler (AMOS) was designed to offer a modular approach to genome assembly. AMOS includes a wide range of tools for assembly, including the lightweight de novo assemblers Minimus and Minimo, and Bambus 2, a robust scaffolder able to handle metagenomic and polymorphic data. This protocol describes how to configure and use AMOS for the assembly of Next Generation sequence data. Additionally, we provide three tutorial examples that include bacterial, viral, and metagenomic datasets with specific tips for improving assembly quality.Curr. Protoc. Bioinform. 33:11.8.1‐11.8.18. © 2011 by John Wiley & Sons, Inc.

Comparative Yersinia genomics identifies features responsible for the colonization of specific host habitats and the horizontal transfer of virulence determinants.

BackgroundThe current revolution in genomics has been made possible by software tools called genome assemblers, which stitch together DNA fragments “read” by sequencing machines into complete or nearly complete genome sequences. Despite decades of research in this field and the development of dozens of genome assemblers, assessing and comparing the quality of assembled genome sequences still relies on the availability of independently determined standards, such as manually curated genome sequences, or independently produced mapping data. These “gold standards” can be expensive to produce and may only cover a small fraction of the genome, which limits their applicability to newly generated genome sequences. Here we introduce a de novo probabilistic measure of assembly quality which allows for an objective comparison of multiple assemblies generated from the same set of reads. We define the quality of a sequence produced by an assembler as the conditional probability of observing the sequenced reads from the assembled sequence. A key property of our metric is that the true genome sequence maximizes the score, unlike other commonly used metrics.ResultsWe demonstrate that our de novo score can be computed quickly and accurately in a practical setting even for large datasets, by estimating the score from a relatively small sample of the reads. To demonstrate the benefits of our score, we measure the quality of the assemblies generated in the GAGE and Assemblathon 1 assembly “bake-offs” with our metric. Even without knowledge of the true reference sequence, our de novo metric closely matches the reference-based evaluation metrics used in the studies and outperforms other de novo metrics traditionally used to measure assembly quality (such as N50). Finally, we highlight the application of our score to optimize assembly parameters used in genome assemblers, which enables better assemblies to be produced, even without prior knowledge of the genome being assembled.ConclusionLikelihood-based measures, such as ours proposed here, will become the new standard for de novo assembly evaluation.

Genes whose expression is growth factor regulated are likely to be important components in the mechanisms controlling cell proliferation and differentiation. With the aim of identifying some of those genes, a lambda cDNA library was prepared with poly(A)+ RNA from quiescent NIH 3T3 cells stimulated with serum for 4 h in the presence of cycloheximide. Differential screening of approximately 200,000 recombinant phage plaques revealed 2,540 clones that cross hybridized preferentially with [32P]cDNA derived from RNA of stimulated cells rather than with cDNA derived from nonstimulated cells. Cross hybridization of these clones identified 82 independent sequences, including c-fos and c-myc. Seventy-one clones were further studied. Analysis of the changes in transcription and mRNA levels after serum stimulation demonstrated that the kinetics and extent of the induction vary dramatically between the different genes. Cycloheximide in all cases superinduced the mRNA levels by two mechanisms, inhibiting the shutoff of transcription and prolonging the half-lives of the mRNAs. Our results showed that induction of proliferation is accompanied by the onset of a complex genetic program.