Robert T. Schimke

A Rapid Chemical Method for Detecting PPLO Contamination of Tissue Cell Cultures

Summary A rapid, simple and routine chemical method for detection of PPLO contamination in cell cultures is reported. The method is based on the presence of arginine deiminase activity in all tissue culture PPLO strains, but absence from all mammalian tissue and PPLO-free cell cultures. In a total of 73 cell cultures examined, the detection of PPLO determined by presence of arginine deiminase activity agreed entirely with the most sensitive cultural procedure which requires 14 days.

[18] Methods for analysis of enzyme synthesis and degradation in animal tissues

Publisher Summary This chapter discusses the methods used for the analysis of enzyme synthesis and degradation in animal tissues. It is well established that the amount of an enzyme present in animal tissues results from opposing processes of continual synthesis and degradation and that either process can be altered by hormonal, nutritional, developmental, or genetic factors. There are a number of methods to assess the contributions of altered synthesis and/or degradation to changes in enzyme (protein) level, including immunologic methods, uses of tracer techniques, and analysis of time courses of changes in enzyme levels. Each method embodies certain assumptions, as well as limitations, and unambiguous conclusions generally can be made only when independent estimates of the rates of synthesis and degradation are made and when more than one method is employed. Tracer techniques are used most commonly for estimating degradation rates. Although such techniques may seem direct, they are full of pitfalls that can result in ambiguous results. Isotope methods can be divided into two general techniques: (1) single isotope administration and (2) continuous isotope administration. The use of reutilizable isotopes in pulse-decay experiments, although commonly employed, has severe limitation(s) in interpretation. They do not, by themselves, provide measures of true half-lives of enzymes. They can, under suitable conditions, provide information on whether two or more proteins in a similar tissue have different rates of turnover.

[64] Assay of ovalbumin mRNA in reticulocyte lysate

Publisher Summary For studies of the regulation of protein synthesis in higher organisms and for the isolation of specific mRNAs, it is necessary to have sensitive and quantitative assays of mRNA content of tissues. This chapter found the crude rabbit reticulocyte lysate system to be an adequate system for such assays. The techniques are simple, and specific protein synthesis directed by exogenous RNA can be monitored readily by use of highly specific immunologic techniques. Although the procedures described in the chapter are for the synthesis of ovalbumin, these techniques work equally well for conalbumin and rat serum albumin mRNAs. Thus the reticulocyte lysate system may provide a general method for the quantitative measure of mRNAs from animal tissue.

TURNOVER OF RAT LIVER ARGINASE

The subject of protein turnover in animal tissues is vast in complexities of subject matter, choices of experimental approach, and problems of interpretation of results. Review papers of Schoenheimer and Rittenberg,’ Tarver,2 and Mandelstam? have indicated many of the problems encountered in the studies of protein turnover, as well as the results obtained. This paper will not attempt to review the entire subject of protein turnover in animal tissues. We shall, rather, limit the discussion of protein turnover to a single animal tissue, namely the liver of the rat, and-specifically-to a single protein, the enzyme arginase. Evidence will be presented that turnover-that is, a continual synthesis and degradation-of this enzyme exists. We shall also examine several different physiological states in which the level of this enzyme changes and shall demonstrate that the rates of synthesis and degradation of this protein can be altered independently to produce the observed changes in arginase levels.

[7] Amplification of genes in somatic mammalian cells

Publisher Summary This chapter describes the experience from the laboratory with techniques employed to generate resistance phenomena as a result of gene amplification. The experience has focused primarily on resistance to methotrexate and amplification of the dihydrofolate reductase gene in Chinese hamster ovary and mouse 3T6 cells. Ones anticipate that experience may prove useful to other investigators working on very different systems. However, it is likely that the mechanisms by which drug treatments alter cell metabolism or kill cells differ with the particular treatment undertaken. The chapter describes the effects of various treatments on cell physiology are critical in studying amplification processes. It emphasizes on the variability of cells in culture, particularly with respect to phenomena such as gene amplification, where the process under investigation necessarily involves genome instability. Even cell lines carded for long periods in the same laboratory without apparent change must be repeatedly subcloned and characterized.

[59] Immunoadsorption of ovalbumin synthesizing polysomes and partial purification of ovalbumin messenger RNA

Publisher Summary An understanding of the mechanisms underlying the regulation of specific protein synthesis requires the ability to isolate and quantitate the various elements that may be rate limiting in specific protein synthesis, including specific mRNA. In special instances a specific mRNA may be isolated because it constitutes such a large portion of the total mRNA of a cell type, and is of a unique size such that it can be separated from other RNA species by size; e.g., globin mRNA in red blood cells, or by virtue of unique physical properties resulting from an unusual base composition, e.g., silk fibroin mRNA. However the majority of mRNAs will require other methods for isolation.