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UNIT 17.6 Detection and Analysis of Proteins Modified by O-linked N-Acetylglucosamine (Natasha Zachara, Robert Cole, and Gerald Hart, Johns Hopkins University Medical School, Baltimore, Maryland; and Yuan Gao, Deakin University, Victoria, Australia). The modification of Ser and Thr residues with O-linked b -N-acetyl glucosamine (O-GlcNAc) is a common and essential modification of nuclear and cytoplasmic proteins, and it is thought that O-GlcNAc performs a regulatory role in the cell. This unit concentrates on the techniques for the detection and analysis of proteins modified by O-GlcNAc as well as methods for the analysis of enzymes responsible for the addition and removal of this group.
UNIT 23.8 Manipulation of the Mouse Genome (David A. Conner,
Harvard Medical School, Cambridge, Massachusetts). This informative unit describes
the management of mouse colonies created by gene targeting. Whether you can
do this in your own lab or work with a core animal facility, you’ll find
this unit very helpful. It defines guidelines necessary to establish the colony,
and describes screening for germline transmission, marking the mice for easy
and consistent identification, and genotyping DNA by PCR or Southern blotting
analysis. It also reviews the software currently available for colony data
management.
UNIT 25B.5 AFLP-based transcript profiling (Pieter Vos and Patrick Stanssens,
Keygene NV, Wageningen, The Netherlands). This unit presents another display
alternative that allows the quantification of transcripts, based on AFLP-fingerprinting
of double-stranded cDNA. The protocol described includes the following steps:
(1) the isolation of poly(A)+ RNA from total RNA, (2) the synthesis of double
stranded cDNA, (3) the preparation of template fragments by digestion of the
cDNA library with a combination of two restriction enzymes and the ligation
of adaptors to the fragment ends, (4) the selective amplification of specific
subsets of fragments, and (5) the electrophoretic analysis of these amplification
products on standard denaturing polyacrylamide gels. The transcript profiles
obtained by this technique are a reliable and efficient tool to identify differentially
expressed mRNAs.
UNIT 25B.6 Serial Analysis of Gene Expression (Seth Blackshaw, Jae B. Kim, Brad St. Croix, and Kornelia Polyak, Harvard Medical School, Boston, Massachusetts). Serial Analysis of Gene Expression (SAGE) involves the generation of short fragments of DNA, or tags, from a defined point in the sequence of all cDNAs in the sample analyzed. This short tag, because of its defined presence in the sequence, is typically sufficient to uniquely identify every transcript in the sample. A comprehensive profile of gene expression in any sample can thus be generated. Absolute, rather than relative, measurements of RNA abundance levels are made, which will be useful in the reliable comparison of data between laboratories.
FORTHCOMING
UNIT 21.7 Chromatin Assembly Using a Drosophila System (Dmitry V. Fyodorov and Mark E. Levenstein, University of California San Diego, La Jolla, California). To successfully study chromatin structure and activity in vitro, it is essential to have a chromatin assembly system that will prepare extended nucleosome arrays with highly defined protein content that resembles bulk chromatin isolated from living cell nuclei in terms of periodicity and nucleosome positioning. The Drosophila ATP-dependent chromatin assembly system described in this unit meets these requirements. The end product of the reaction described here has highly periodic extended arrays with physiologic spacing and positioning of the nucleosomes.
UNIT 25A.2 Preparation of Single Cells from Solid Tissues for Analysis by PCR (Nancy Sawtell, Children’s Hospital Medical Center, Cincinnati, Ohio). This unit details a protocol for the separation of solid tissues into single cell suspensions for subsequent analysis of nucleic acids and protein. Tissues are fixed in situ by perfusion, terminating cell processes and thus changes that would accompany dissociating the living tissue. The procedure is particularly useful when the cell type of interest represents a minor population relative to other cells types in the tissue. Once separated, individual cells or groups of a particular cell type can then be analyzed using PCR strategies. The procedure can also be adapted to allow the quantification of the number of cells within a tissue containing specific nucleic acid sequences, for example, a particular viral DNA or RNA sequence.
APPENDIX 1H Safe Use of Hazardous Chemicals (George Lunn, Baltimore, Maryland, and Gretchen Lawler, Purdue University, West Lafayette, Indiana). Performing some of the experiments in Current Protocols may result in exposure to toxic chemicals or carcinogenic, mutagenic, or teratogenic agents. We assume that the researchers using these procedures would be aware of necessary safety considerations, but we offer specific guidelines and cautionary notes wherever possible throughout the manual. This appendix unit is also a good source of information about precautions necessary for good laboratory practice. In this update, we provide information on the most commonly used hazardous chemicals, examples of chemical incompatibility, chemical resistance of commonly used brands of gloves, and methods for detection, decontamination, and disposal of many hazardous substances. New procedures on handling enzyme inhibitors are also included. We guarantee that this unit will be often used.
OTHER TOPICS IN DEVELOPMENT
Lentiviral system
Microarray data analysis
Representational Difference Analysis