High-Density Sequencing Applications in Microbial Molecular Genetics, Volume 612 in the Methods of Enzymology series provides the latest on the high-density sequencing of DNA and cDNA libraries and how they have revolutionized contemporary research in biology. Methods permitting tens of millions of sequence reads in a single experiment have paved the way to genome-wide studies that are contributing to our understanding of the complexity of living systems. Chapters in this updated volume include Characterizing the role of exoribonucleases in the control of microbial gene expression: Differential RNA seq., Conformational studies of bacterial chromosomes by high-throughput sequencing methods, Measuring mRNA degradation, and more.
Addition sections cover Global recognition patterns of bacterial RNA-binding proteins, High-resolution profiling of NMD targets, and the Generation of a metagenomic 3C/Hi-C library of human gut microbiota, Genome-wide mapping of yeast retrotransposons integration target sites, Measuring protein synthesis rates, Finding unsuspected partners of small RNAs with new screening approaches, Use of multiplexed transcriptomics to define the relationship between promoter sequence and transcription output, RNA-based control of quorum sensing in Vibrio cholerae, amongst other highly regarded topics.
- Detail methods used in research articles that were recently published in leading journals
- Provides the latest on the high-density sequencing of DNA and cDNA libraries and how they have revolutionized contemporary research in biology
Students and researchers interested in using high-density sequencing methods as a tool for exploring the complexity of living systems. In addition to detailing protocols for a variety of applications in microbial molecular genetics, this volume will help a new generation of researchers to innovate by applying this technology to their research projects
Agamemnon James (A.J.) Carpousis is a Research Director in the CNRS. He graduated with honors in Biochemistry from the University of Pennsylvania and then did his doctoral studies in the Molecular Biology program at UCLA. His PhD work was on the mechanism of transcription initiation by Escherichia coli RNA polymerase. After postdoctoral research at UC Santa Barbara and the University of Geneva, A.J. Carpousis joined the LMGM, which is a CNRS Microbial Molecular Genetics Laboratory at the University of Toulouse. His research in Geneva contributed to the discovery that RNase E, which is an essential ribonuclease in E. coli, is a key enzyme in the initiation of mRNA degradation. In subsequent research, he purified RNase E and showed that it associates with other proteins involved in mRNA degradation forming a multienzyme complex, which is now known as the RNA degradosome. His group in Toulouse showed that RNase E has a composite structure consisting of a catalytic domain and a large non-catalytic region that serves as the scaffold for interactions with other components of the RNA degradosome. Other work includes studies on the role of RhlB, PNPase and poly(A) polymerase in mRNA degradation, and identification and characterization of beta-CASP ribonucleases in the Archaea. More recently, A.J. Carpousis and his colleagues showed that the RNA degradosome is localized to the inner cytoplasmic membrane of E. coli. They characterized a conserved element in the non-catalytic region of RNase E that directly anchors the RNA degradosome to the phospholipid bilayer of the inner membrane. RNA degradosomes on the inner membrane are highly dynamic forming short-lived clusters that are hypothesized to be centers of mRNA degradation. His group currently uses molecular genetics, biochemistry, high-density sequencing methods and super-resolution microscopy to address the question of the composition and supramolecular structure of the RNA degradosome clusters.