Michael M. Crerar

Quantitation of muscle glycogen phosphorylase mRNA and enzyme amounts in adult rat tissues

Mammalian glycogen phosphorylases comprise a family of isozymes that are expressed selectively in a variety of cell types. As an initial step towards understanding the molecular processes that regulate the differential expression of the phosphorylase family, we have begun a quantitative examination of isozyme expression in vivo. In this paper, we report quantitative estimates of the amounts of the muscle (M) isozyme and its mRNA in adult rat tissues. Quantitative estimates of the amount of M-phosphorylase were obtained by an analysis involving electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose filters and sequential treatment with M-isozyme specific antibody and radioactively- labeled protein A. M-phosphorylase mRNA amounts were determined by an analysis involving transfer of RNA from agarose gels to nitrocellulose filters and subsequent hybridization with radioactively labelled rat M-phosphorylase cDNA. These studies indicate that M-phosphorylase is present in all tissues tested with the possible exception of liver. These are skeletal muscle, heart, brain, stomach, lung, kidney, spleen and testis. Quantitation of M-phosphorylase amounts indicate that there is a wide spectrum of variation (over 1000-fold range) in the relative amounts of the M-isozymes in these tissues. Relative mRNA levels parallel isozyme levels indicating that the major control of expression of this isozyme is governed by mRNA accumulation.

Comparative analysis of species-independent, isozyme-specific amino-acid substitutions in mammalian muscle, brain and liver glycogen phosphorylases

Mammalian glycogen phosphorylases exist as three isozymes, muscle, brain and liver, that exhibit different responses to activation by phosphorylation and AMP, regardless of species. To identify species-independent, amino-acid substitutions that may be important determinants in differential isozyme control, we have sequenced cDNAs containing the entire protein coding regions of rat muscle and brain phosphorylases. Nucleotide sequence comparisons with rat liver, rabbit muscle, and human muscle, brain and liver phosphorylase genes, indicate that muscle and brain isozymes are more related to each other than to the liver isozyme. Unlike the human isozymes, there is little difference in GC content of codons in the rat isozymes. In relation to the rabbit muscle isozyme three-dimensional structure, amino-acid sequence comparisons indicate that very few nonconservative isozyme-specific substitutions occur in buried and dimer contact residues. There is strict conservation of active site, pyridoxal-phosphate-binding site and nucleoside inhibitor site residues, as well as CAP loop and helix-2 residues that comprise the phosphorylation activation and part of the AMP binding sites. In contrast, five liver isozyme-specific substitutions occur between residues 313-325 and another at residue 78 which may be important determinants in the poor activation of this isozyme by AMP. Substitutions in the brain isozyme at residues 21-23, 405 and 435 may play a role in its poor response to activation by phosphorylation.

DNA polymerase activity from Tetrahymena pyriformis

The DNA polymerase of Z’etrahymena pyrifomis was first isolated from crude extracts of the cells by Pearlman and Westergaard [ 1,2] . The specific activity of this polymerase was increased if, prior to extraction, the cells were treated with either ultraviolet irradiation, electron irradiation, methotrexate plus uridine or ethidium bromide [2&4]. Using Sephadex G-200 column chromatography in 0.5 M NaCl of crude extracts of Tetrahymena, two DNA polymerase activities were observed after ultraviolet irradiation, electron irradiation or methotrexate plus uridine treatment [3] . The enzyme fraction showing increase in specific activity was found in the mitochondria [4] . As a first step in asking questions about the relation of “nuclear” to mitochondrial DNA polymerases and about the enzymes involved in DNA replication and DNA repair [2,5] we have devised a fractionation scheme to partially purify the DNA polymerase from untreated exponentially growing cells. Using this fractionation scheme, we have attempted to fractionate the activity from ethidium bromide treated cells. Our fractionation method was based on an observation made with the DNA polymerases from sea urchin embryos [6] and yeast [7] and observed independently by us. Under conditions of low salt, the DNA polymerase aggregates into high molecular weight species, and in high salt dissociates into a low molecular weight species. This characteristic of the enzyme has enabled us to purify the enzyme approximately 85 fold. Part of the increase in specific activity in ethidium bromide treated cells is detected as a new activity peak when chromatographed in low salt on Sephadex G-200.

The genes for ?-myosin heavy chain and glycogen phosphorylase are discoordinately regulated during compensatory growth of plantaris muscle in the adult rat

It has been shown previously that compensatory hypertrophy of the plantaris muscle in adult rats can be induced by surgical removal of the synergistic gastrocnemius muscle. During hypertrophy, muscle transformation also occurs and there is a shift in the fiber type population of the muscle from fast to slow. Towards obtaining a better understanding of the molecular mechanisms controlling this process, we have carried out a kinetic analysis of the change in expression of two muscle-specific genes encoding the slow β-heavy chain isoform of myosin and the muscle isoform of glycogen phosphorylase. This analysis indicated that significant increases (2–3 fold) in the steady-state levels of slow myosin heavy chain mRNA and protein did not occur until several weeks following ablation of the gatrocnemius muscle. Increases in slow fiber type paralleled the change in β-myosin heavy chain expression. In contrast, the activity of phosphorylase, as well as the level of its corresponding mRNA, decreased approx. 1.5-2 fold shortly after (2-4 days) ablation of the gastrocnemius and levels remained low for at least several weeks. Significant changes in expression of these genes did not occur in plantaris muscle from sham operated contralateral legs. These studies indicated that changes in the expression of both genes was governed primarily by accumulation of their mRNAs. However, these genes were not coordinately regulated, indicating either that multiple control mechanisms regulate gene expression in this system or that the same controlling factor(s) regulates expression of these genes in temporally different ways.

Chapter 17 Manipulations with Tetrahymena pyriformis on Solid Medium

Publisher Summary This chapter discusses the two methods for growing and cloning Tetrahymena on solid medium on petri plates. The first method involves the use of Sephadex beads on the plates to aid in restricting the movement of cells while still allowing them to remain motile and divide. The Sephadex beads on the plates aid in restricting the movement of cells while still allowing them to remain motile and divide. Most of the liquid in which cells are added to the plate is taken up by the agar and by the Sephadex, and spaces between Sephadex beads remain dry. Thus Tetrahymena cannot migrate between Sephadex beads and single cells are localized around beads in a liquid environment with sufficient nutrients to allow for motility, growth, and division. The second method involves growth of Tetrahymena in micro-holes punched in the surface of agar medium. Cells can be grown, cloned, and replica plated using this method. Thus, these methods should be very valuable in some of the steps in mutant isolation and genetic manipulation in Tetrahymena.