Methods in Cell Biology 

About: Methods in Cell Biology is an academic journal published by Elsevier BV. The journal publishes majorly in the area(s): Biology & Zebrafish. It has an ISSN identifier of 0091-679X. Over the lifetime, 3676 publications have been published receiving 120669 citations. The journal is also known as: Methods Cell Biol.

Preparation of isolated rat liver cells.

Abstract: Publisher Summary This chapter discusses preparation of isolated rat liver cells The early mechanical and chemical methods for liver-cell preparation were relatively successful in converting liver tissue to a suspension of isolated cells The successful preparation of intact liver cells by perfusion with collagenase is technically quite difficult The major method for preparation of nonparenchymal liver cells is based on the selective sensitivity of parenchymal cells toward proteases By incubation of rat liver minces with pronase, most of the liver parenchyma is digested, while nonparenchymal cells remain intact and can be recovered from the incubate Similar results have been reported with trypsin digestion of collagenase-dispersed liver minces, but pronase appears to be more effective The most common procedure is to perfuse the liver briefly with pronase before it is minced and incubated with the enzyme Such direct pronase methods have been used by several investigators with yields of nonparenchymal liver cells reported to be in the range 2–15 × 10 6 cells/gm liver

Transcription and translation.

Abstract: Publisher Summary This chapter reviews the current state of the knowledge of transcription and translation in Caenorhabditis elegans. Transcription in eukaryotes is accomplished by three RNA polymerases, each transcribing a particular type of RNA; RNA polymerase I transcribes ribosomal RNA, RNA polymerase II transcribes mRNAs and snRNAs, and RNA polymerase III transcribes tRNAs and other low-molecular-weight RNAs. The chapter focuses on RNA polymerase II transcription. The C. elegans gene for the large subunit of RNA polymerase II has been cloned, and has shown to encode a protein that resembles its vertebrate counterparts. For most polymerase II transcribed genes in C. elegans, a sequence fitting the consensus TATA element about 30 bp upstream of the transcriptional start site is found. Recently, a cDNA encoding the TATA box binding protein, or TATA binding protein (TBP) subunit of transcription factor II D (TFIID), has been identified in the nematode. This protein shows extended regions of sequence similarity to vertebrate TBP, binds to a TATA box sequence and can substitute for vertebrate TFIID basal activity in in vitro extracts. These results illustrate a high degree of evolutionary conservation in the basic transcription machinery.

Chapter 19 DNA Transformation

Abstract: Publisher Summary This chapter discusses DNA transformation. DNA transformation assays in a whole organism provide experimental links between molecular structure and phenotype. Experiments with transgenic Caenorhabditis elegans start in general with the injection of DNA into the adult gonad. Effects on phenotype or gene expression patterns can be analyzed either in F1 progeny derived from the injected animals or in derived transgenic lines. Germ-line transformation has been achieved by microinjection of DNA directly into oocyte nuclei or by microinjection of DNA into the cytoplasm of the hermaphrodite syncytial gonad. Three forms of heritable DNA transformation have been observed in C. elegans are: (1) extra chromosomal transformation; (2) non-homologous integration; and (3) homologous integration. Setting up microinjection in a laboratory already equipped for C. elegans genetics and molecular biology requires a modest investment in space and money. A separate easily scoreable marker gene to identify transformed animals can be extremely useful in a variety of injection experiments. The propensity for injected DNA molecules to recombine with each other generally allows one to coinject the selectable marker with a DNA segment to be tested for activity.

Cell cycle extracts.

Abstract: Publisher Summary This chapter reviews the early Xenopus cycle and the history of cell cycle extracts. The early Xenopus cell cycles are controlled by the activation and inactivation of a protein kinase named maturation-promoting factor (MPF). MPF is also known as the cdc2 kinase and growth-associated histone H1 kinase, and consists of two principal subunits, the catalytic p34cdc2 subunit and cyclin. Activation of MPF induces both mitosis and meiosis, whereas its inactivation triggers the onset of anaphase and the progression into interphase. The fluctuation of MPF activity in early Xenopus embryos is discussed in the chapter. The chapter discusses the methods for preparing and utilizing frog cell cycle extracts. Female frogs are primed for ovulation by injecting them with pregnant mare serum gonadotropin (PMSG) and then induced to ovulate by a subsequent injection of human chorionic gonadotropin (HCG). The chapter also describes sperm nucleus preparation.