Myophosphorylase gene transfer in McArdle's disease myoblasts in vitro.

McArdle's disease is due to a genetic deficiency of glycogen phosphorylase and results in a lack of glucose mobilization from glycogen during anaerobic exercise. A genetic defect in Merino sheep produces a similar picture. We constructed a first-generation adenoviral recombinant containing the full-length human phosphorylase cDNA under the control of the Rous sarcoma virus promoter. Primary myoblast cultures from phosphorylase-deficient human and sheep muscle were efficiently transduced with this vector, resulting in restoration of the phosphorylase activity. A similar correction of the genetic defect in muscles of McArdle's patients in vivo appears feasible, preferably with the use of an adeno-associated viral vector.

Chimeric muscle and brain glycogen phosphorylases define protein domains governing isozyme-specific responses to allosteric activation.

Muscle and brain glycogen phosphorylases differ in their responses to activation by phosphorylation and AMP. The muscle isozyme is potently activated by either phosphorylation or AMP. In contrast, the brain isozyme is poorly activated by phosphorylation and its phosphorylated a form is more sensitive to AMP activation when enzyme activity is measured in substrate concentrations and temperatures encountered in the brain. The nonphosphorylated b form of the brain isozyme also differs from the muscle isozyme b form in its stronger affinity and lack of cooperativity for AMP. To identify the structural determinants involved, six enzyme forms, including four chimeric enzymes containing exchanges in amino acid residues 1-88, 89-499, and 500-842 (C terminus), were constructed from rabbit muscle and human brain phosphorylase cDNAs, expressed in Escherichia coli, and purified. Kinetic analysis of the b forms indicated that the brain isozyme amino acid 1-88 and 89-499 regions each contribute in an additive fashion to the formation of an AMP site with higher intrinsic affinity but weakened cooperativity, while the same regions of the muscle isozyme each contribute to greater allosteric coupling but weaker AMP affinity. Kinetic analysis of the a forms indicated that the amino acid 89-499 region correlated with the reduced response of the brain isozyme to activation by phosphorylation and the resultant increased sensitivity of the a form to activation by saturating levels of AMP. This isozyme-specific response also correlated with the glycogen affinity of the a forms. Enzymes containing the brain isozyme amino acid 89-499 region exhibited markedly reduced glycogen affinities in the absence of AMP compared to enzymes containing the corresponding muscle isozyme region. Additionally, AMP led to greater increases in glycogen affinity of the former set of enzymes. In contrast, phosphate affinities of all a forms were similar in the absence of AMP and increased approximately the same extent in AMP. The potential importance of a number of isozyme-specific substitutions in these sequence regions is discussed.

Evolution of allosteric control in glycogen phosphorylase.

In relation to the primary sequence and three-dimensional structure of rabbit muscle glycogen phosphorylase, we have carried out a comparative sequence analysis of phosphorylases from human, rat, Dictyostelium, yeast, potato and Escherichia coli. Based on sequence similarity, a large region of the protein is shared by these enzymes extending from alpha-helix-1 to the last alpha-helix-33. Conserved residues are equally distributed between the N and C-terminal domains and occur primarily in buried residues. Phylogenetic analysis indicates that the two isozymes within either E. coli, potato or Dictyostelium are more closely related to each other than they are to other phosphorylases. Yeast phosphorylase is most closely related to the Dictyostelium isozymes. Mammalian muscle and brain isozymes are more closely related to each other than to the liver isozyme and the muscle isozyme is evolving at the slowest rate. All phosphorylases exhibit high conservation of active site and pyridoxal phosphate binding residues. Most phosphorylases also exhibit high conservation of sugar binding residues in the glycogen storage site. Phosphorylation and AMP binding site residues are poorly conserved in non-mammalian phosphorylases. In contrast, glucose-6-P binding residues are highly conserved in four of the seven non-mammalian enzymes. Analysis of interacting pairs of dimer contact residues indicates that they can be grouped into three relatively independent networks. One network contains phosphorylation and AMP binding residues and is poorly conserved in non-mammalian enzymes. A second network contains glucose-6-P binding residues and is highly conserved in enzymes containing a conserved glucose-6-P binding site. A third, conserved network contains residues within the tower helix and gate loop. A model for the evolution of allostery in phosphorylase is proposed, suggesting that glucose-6-P inhibition was an early control mechanism. The later creation of primarily distinct ligand binding sites for AMP/phosphorylation control may have allowed the establishment of a separate dimer contact network for propagating conformational changes leading to activation rather than inhibition of enzyme activity.

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.

Glucose and caffeine regulation of liver glycogen phosphorylase activity in the freeze-tolerant wood frog Rana sylvatica.

We have examined the effect of glucose and caffeine inhibition on the activity of liver glycogen phosphorylase a from the freeze-tolerant frog Rana sylvatica. Kinetic studies indicate that this enzyme exhibits similar sensitivity to glucose inhibition (glucose dissociation constant = 12.5 mM) as the mammalian enzyme. Little inhibition (less than 25%) was observed at normal glucose concentrations (1-5 mM), while significant inhibition (60-95%) occurred at glucose concentrations (50-500 mM) present in freezing-exposed animals. These results favour the hypothesis that in the normal state glucose regulates phosphorylase activity primarily through the promotion of dephosphorylation of phosphorylase a, whereas during freezing regulation is achieved through phosphorylase a inactivation. The caffeine dissociation constant (0.93 mM) and the degree of synergism between caffeine and glucose (interaction factor, alpha = 0.14) were also similar to that observed for the mammalian enzyme. Hence, if a caffeine-like ligand exists in vivo, it must be in low enough amounts during freezing to allow sufficient phosphorylase a activity for high glucose production.

Localization of the muscle, liver, and brain glycogen phosphorylase genes on linkage maps of mouse chromosomes 19, 12, and 2, respectively.

Mammalian glycogen phosphorylases comprise a family of three isozymes, muscle, liver, and brain, which are expressed selectively and to varying extents in a wide variety of cell types. To better understand the regulation of phosphorylase gene expression, we isolated partial cDNAs for all three isozymes from the rat and used these to map the corresponding genes in the mouse. Chromosome mapping was accomplished by comparing the segregation of phosphorylase restriction fragment length polymorphisms (RFLPs) with 16 reference loci in a multipoint interspecies backcross between Mus musculus domesticus and Mus spretus. The genes encoding muscle, liver, and brain phosphorylases (Pygm, Pygl, and Pygb) are assigned to mouse chromosomes 19, 12, and 2, respectively. Their location on separate chromosomes indicates that distinct cis-acting elements govern the differential expression of phosphorylase isozymes in various tissues. Our findings significantly extend the genetic maps of mouse chromosomes 2, 12, and 19 and can be used to define the location of phosphorylase genes in man more precisely. Finally, this analysis suggests that the previously mapped "muscle-deficient" mutation in mouse, mdf, is closely linked to the muscle phosphorylase gene. However, muscle phosphorylase gene structure and expression appear to be unaltered in mdf/mdf mice, indicating that this mutation is not an animal model for the human genetic disorder McArdle's disease.

The genes for beta-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 beta-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 beta-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.

Studies on the expression and evolution of the glycogen phosphorylase gene family in the rat.

Muscle, liver, and brain glycogen phosphorylases in mammals comprise a family of closely related isozymes that are differentially expressed in a wide variety of cell types. Towards obtaining a better understanding of the mechanisms governing the tissue-specific control of expression of this isozyme family, we used an antibody generated against bovine liver phosphorylase to obtain quantitative estimates of the concentrations of the three isozymes in rat tissues by Western blot analysis. This analysis indicated that expression of these isozymes at the protein level, although widespread, was tissue-specific and each isozyme exhibited variations in expression throughout the tissues where it was produced. We also began a preliminary analysis of the evolution of the genes encoding these three isozymes. Towards this end, we isolated and sequenced a partial cDNA to the rat brain isozyme that encompassed the coding region from amino acids 569 to 729. Using known phosphorylase gene sequences, we reconstructed a phylogeny spanning three kingdoms. This phylogeny indicated that brain and muscle isozymes are more closely related to each other than to the liver isozyme and that gene duplications that give rise to the family predate the mammalian radiation. Differences in the relative rates of change of the three isozymes were observed and this may reflect different constraints on their evolution perhaps related to their functional roles and (or) tissue-specific expression.

Electrophoretic analysis of liver glycogen phosphorylase activation in the freeze-tolerant wood frog.

As an adaptation for overwinter survival, the wood frog, Rana sylvatica is able to tolerate the freezing of extracellular body fluids. Tolerance is made possible by the production of very high amounts of glucose in liver which is then sent to other organs where it acts as a cryoprotectant. Cryoprotectant synthesis is under the control of glycogen phosphorylase which in turn is activated in response to ice formation. To determine the mechanism of phosphorylase activation, a quantitative analysis of phosphorylase protein concentration and enzymatic activity in liver was carried out following separation of the phosphorylated a and nonphosphorylated b forms of the enzyme on native polyacrylamide gels. The results suggest that in gels, the b form is completely inactive, even in the presence of AMP and sodium sulfate, whereas the a form is active and stimulated 3-fold by these substances. Further, phosphorylase activation appears to arise solely from conversion of the b to a form of the enzyme without an increase in phosphorylase concentration or activation of a second isozyme. The quantitative analysis presented here should prove generally useful as a simple and rapid method for examining the physiological and genetic regulation of phosphorylase in animal cells.

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 sequence analysis of rat, rabbit, and human muscle glycogen phosphorylase cDNAs.

As an initial step in the investigation of the structure, evolution and developmental regulation of the glycogen phosphorylase gene family, we have isolated partial cDNAs to rat, rabbit and human muscle phosphorylase mRNAs. Sequence comparisons of these cDNAs in regions that encode portions of the enzyme located near and encompassing the C terminus show that there is a high degree of interspecies conservation of structure in this region. Conservation of amino acid and nucleotide sequence is high, approximately 96% and 90% homology, respectively, among all three species. In addition, most of the amino acid changes that have occurred conserve the chemical nature of the amino acid side-chains affected. The changes can be easily accommodated in the rabbit muscle phosphorylase tertiary structure and appear to have little effect on the overall conformation. Interestingly the rat and human enzymes lack the carboxyl-terminal proline (residue 841) present in the rabbit enzyme and terminate at isoleucine (residue 840). The genetic basis for this difference in carboxyl termini is unknown. However, unlike the other amino acid changes, it cannot be accounted for by a single base-pair substitution. A comparison of the 3' untranslated regions in these cDNAs shows that there has been little constraint on the evolutionary divergence of most of this region (70% homology among the three species). There are, however, two repeated segments of DNA flanking the stop codons that are identical among all three species. Similar sequences are found within regions of DNA that contain a variety of transcriptional enhancers, suggesting the possibility that the repeats may be functional.

Myogenic differentiation of L6 rat myoblasts: evidence for pleiotropic effects on myogenesis by RNA polymerase II mutations to alpha-amanitin resistance.

To assess the functional role of RNA polymerase II in the regulation of transcription during muscle differentiation, we isolated and characterized a large number of independent alpha-amanitin-resistant (AmaR) mutants of L6 rat myoblasts that express both wild-type and altered RNA polymerase II activities. We also examined their myogenic (Myo) phenotype by determining their ability to develop into mature myotubes, to express elevated levels of muscle creatine kinase, and to synthesize muscle-characteristic proteins as detected by two-dimensional polyacrylamide gel electrophoresis. We found a two- to threefold increase in the frequency of clones with a myogenic-defective phenotype in the AmaR (RNA polymerase II) mutants as compared to control ethyl methane sulfonate-induced, 6-thioguanine-resistant (hypoxanthine, guanine phosphoribosyl transferase) mutants or to unselected survivors also exposed to ethyl methane sulfonate. Subsequent analysis showed that about half of these myogenic-defective AmaR mutants had a conditional Myo(ama) phenotype; when cultured in the presence of amanitin, they exhibited a Myo- phenotype; in its absence they exhibited a Myo+ phenotype. This conditional Myo(ama) phenotype is presumably caused by the inactivation by amanitin of the wild-type amanitin-sensitive RNA polymerase II activity and the subsequent rise in the level of mutant amanitin-resistant RNA polymerase II activity. In these Myo(ama) mutants, the wild-type RNA polymerase II is normally dominant with respect to the Myo+ phenotype, whereas the mutant RNA polymerase II is recessive and results in a Myo- phenotype only when the wild-type enzyme is inactivated. These findings suggest that certain mutations in the amaR structural gene for the amanitin-binding subunit of RNA polymerase II can selectively impair the transcription of genes specific for myogenic differentiation but not those specific for myoblast proliferation.

Isolation and characterization of a rat amylase gene family.

Portions of at least nine distinct rat amylase genes or pseudogenes have been isolated. Cloned rat genomic DNA fragments containing complete or major portions of seven of these have been examined by heteroduplex analysis and fall within two separate groups based on their degree of homology. Four gene sequences comprising one of these groups are closely related to pancreatic amylase mRNA. The other group shows significant nonhomology to both pancreatic and parotid amylase cDNAs and may represent an additional gene type(s). All of the cloned amylase gene sequences are found in rat genomic DNA. Additional amylase sequences which have not yet been cloned are also detected. Comparison of DNA from individual Sprague-Dawley rats by Southern blotting techniques indicates allelic variation at multiple amylase loci.

Pancreas-specific genes: structure and expression.

Via recombinant DNA technology the mRNA sequence of pancreatic amylase has been cloned and its nucleotide sequence has been determined. The cloned sequence represents 96% of the total length of amylase mRNA; missing are an estimated 75 +/- 30 nucleotides from the 5' end. The amino acid sequence of rat pancreatic amylase was deduced solely from the nucleotide sequence of the mRNA. Unlike other eukaryotic mRNAs, the amylase mRNA has short 5' and 5' untranslated regions, suggesting that long untranslated regions of eukaryotic mRNAs either do not contain extensive functional sequences or that these sequences are incorporated within the amino acid coding region of amylase mRNA. The cloned amylase mRNA sequence was radiolabeled and used as a probe for in situ hybridization. These experiments demonstrate that amylase mRNA is present in all acinar cells but not in other pancreatic cell types. Using the cloned amylase mRNA sequences as a hybridization probe, three nonoverlapping genomic DNA fragments containing amylase gene sequences were isolated. From the similar sequence organization of the three amylase genes visualized by DNA heteroduplex mapping, a consensus structure of a rat amylase gene is proposed. It is an extended gene structure 10 kilobase pairs in length containing the 1547 base pairs of the cloned mRNA coding sequence interrupted by seven intervening sequences ranging from 400-2000 base pairs long. Thus, in nuclear DNA the amylase mRNA coding sequence is disrupted into at least eight segments from 150-300 base pairs long.

Structure of a family of rat amylase genes.

The sequences of two cloned rat pancreatic amylase cDNAs comprising 95% of the mRNA sequence are reported. Analysis of cloned rat genomic DNA fragments using cloned cDNA probes indicates that the rat genome contains multiple closely related amylase genes in which the cDNA sequences are distributed within a region 9 kilobases in length and are interrupted by at least seven intervening sequences.

Regulation of RNA polymerase II activity in alpha-amanitin-resistant CHO hybrid cells.

CHO hybrid cell lines obtained by fusing cells of wild-type sensitivity to alpha-amanitin with mutant cells containing RNA polymerase II activity resistant to alpha-amanitin have both sensitive (wild-type) and resistant forms of RNA polymerase II. When these hybrids were grown in medium containing alpha-amanitin, the sensitive form of polymerase II was inactivated, and the activity resistant to alpha-amanitin increased proportionally. The total polymerase II activity level therefore remained constant. This regulation of RNA polymerase II activity occurred independently of that of RNA polymerase I and was similar to that observed previously in the alpha-amanitin-resistant rat myoblast mutant clone Ama102 (Somers, Pearson, and Ingles, 1975a). A sensitive radioimmunoassay was developed to quantitate the total mass of RNA polymerase II enzyme. Under conditions of regulation of the enzymatic activity when hybrids grown in alpha-amanitin exhibited a 2-3 fold increase in the activity of the alpha-amanitin-resistant enzyme, no major change in the enzyme mass was detected immunologically. However, quantitation of the alpha-amanitin-inactivated polymerase II of wild-type sensitivity by 3H-amanitin binding indicated that the loss of its enzymic activity was accompanied by a loss of 3H-amanitin binding capacity in the cell lysates. All these results taken together indicate that a mechanism for regulating the intracellular level of RNA polymerase II exists and that it involves changes in the concentration of enzyme.