Products Of Separate Genes Research Articles

Chemical and serologic definition of two unique D region-encoded molecules in the wild-derived mouse strain B10.GAA37.

Detailed serologic and biochemical characterization of D region products of the wild-derived mouse strain B10.GAA37 (Dw16) were performed and compared with previous studies of the D region products of the H-2d,b, and q haplotypes. Serologic analysis revealed that the antigens encoded by the Dw16 region express a unique combination of specificities defined by monoclonal antibodies (mAb) with established activity for the Ld and Dd molecules. Two out of five anti-Ld-reactive mAb reacted with B10.GAA37 cells, whereas one of three anti-Dd mAb showed B10.GAA37 reactivity. Sequential immunoprecipitation of B10.GAA37 antigens demonstrated the existence of at least two antigenically distinct molecules (designated Dw16 and Lw16) encoded by genes associated with the Dw16 region. Peptide map comparisons of the Dw16 and Lw16 molecules defined multiple differences in their primary protein structure, suggesting they are products of separate genes. Structural comparisons of the Lw16 and Dw16 molecules with the Ld and Dd molecules implied a) that the Dw16 and Dd regions did not result from a recent evolutionary divergence of a common primordial haplotype, and b) that the Lw16 and Dw16 molecules are more structurally homologous to each other than the Ld and Dd molecules are. Comparison of these findings with our previous studies of antigens encoded by the D regions suggest that each of these haplotypes has unique properties in terms of the number of gene products expressed and/or the structural relatedness of products of the same region.

Acanthamoeba myosin IA, IB, and II heavy chains are synthesized in vitro from Acanthamoeba messenger RNA.

Addition of a myosin IB polyclonal antibody, which reacts with the heavy chains of both myosins IA and IB, to the [35S]methionine-labeled proteins synthesized in vitro using Acanthamoeba messenger RNA specifically immunoprecipitated radioactive polypeptides that migrated on sodium dodecyl sulfate-polyacrylamide gels exactly with the heavy chains of purified myosins IA and IB. A myosin II polyclonal antibody specifically immunoprecipitated a radioactive polypeptide which migrated exactly with the heavy chain of myosin II. These results provide strong evidence that Acanthamoeba myosins IA, IB, and II are separate gene products and that the purified proteins contain native, undegraded heavy chains. These results are especially important for myosins IA and IB, because they possess unusually small heavy chains for myosins.

The Yc and Ya subunits of rat liver glutathione S-transferases are the products of separate genes.

Rat liver glutathione S-transferases consist of binary combinations of three major classes of subunits designated as Ya (Mr = 25,600), Yb (Mr = 27,000) and Yc (Mr = 28,000). We have determined the nucleotide sequences of a cDNA insert in pGTR262 containing partial sequence of a Yc subunit. Sequence comparison with a Ya subunit cDNA clone pGTR261 revealed 70% nucleotide sequence homology and 65% amino acid sequence homology in the overlapping coding regions. Approximately 65% of the amino acid substitutions between these two subunits occur in clusters of two to eight residues. The 3' noncoding sequences of these two subunit cDNA clones are highly divergent in length and in sequences. The 3' noncoding region of pGTR262 cDNA contains open reading frames of 23, 39, and 13 amino acids which are in phase with the deduced Yc subunit sequences but interrupted by termination codons. By RNA blot hybridization analysis, we found that this Yc subunit sequence expression is tissue specific. It is expressed at a reduced level in kidney and testis as compared with liver and hardly at all in heart, lung, seminal vesicles, and spleen. We suggest that the mechanism for the tissue-specific expression of the Yc subunit of rat liver glutathione S-transferases may occur at or prior to the level of RNA processing. From the divergent DNA sequences between the Ya and Yc subunits reported in this communication and their differential induction upon acute phenobarbital administration reported by Pickett et al. (Pickett, C.B., Donohue, A. M., Lu, A. Y. H., and Hales, B. F. (1982) Arch. Biochem. Biophys. 215, 539-543) we propose that the Ya and Yc subunits of rat liver glutathione S-transferases are encoded by separate gene families or transcriptional units.

Characterization of genomic clones specifying rabbit alpha- and beta-ventricular myosin heavy chains.

We have isolated gene sequences coding for the alpha- and beta-myosin heavy chains (HC) of rabbit ventricular muscle. A rabbit genomic library was screened with previously characterized cDNA clones specifying part of the light meromyosin and the entire subfragment 2 portion of alpha- and beta-myosin HCs, as well as with a clone containing the 3' nontranslated sequences of the alpha-myosin HC mRNA. Seven strongly hybridizing clones were analyzed in detail. One genomic clone encoded all of the 3' nontranslated sequences of an alpha-cDNA clone and, therefore, contained the 3' end of the alpha-myosin HC gene. Electron microscopic heteroduplex analysis and DNA sequence analysis showed that this clone overlapped a second genomic clone providing more than 25 kilobase pairs of the alpha-myosin HC gene. The exons within this region corresponded to approximately equal to 85% of the mRNA and were separated by at least 28 introns. A clone for the beta-myosin HC gene was also identified by Southern blot hybridization, by heteroduplex mapping, and by comparing the DNA sequence of a subfragment 2 exon to sequences of the alpha- and beta-cDNA clones. The introns of the alpha- and beta-myosin HC genes were in the same position but showed marked variation in length. These results conclusively showed that the alpha- and beta-myosin HCs are products of separate genes.

Heterogeneity among human collagenases demonstrated by monoclonal antibody that selectively recognizes and inhibits human neutrophil collagenase.

The heterogeneity of human collagenases has been examined using a monoclonal antibody to neutrophil collagenase. This antibody inhibited collagenase activity and, when covalently coupled to Sepharose, bound both latent and active enzyme. Although human neutrophil collagenase was inhibited by the antibody, the activity of human skin and rheumatoid synovial collagenase was not significantly diminished in the presence of the antibody. Competitive inhibition studies also differentiated between these collagenases. Only human neutrophil collagenase effectively blocked the antibody in a competitive enzyme-linked immunosorbent assay while skin and rheumatoid synovial collagenase again failed to interact with the antibody. The unequivocal recognition of neutrophil collagenase as an immunologically distinct entity from other collagenases supports the hypothesis that neutrophil collagenase is a separate gene product from fibroblast or synovial collagenase.

An electrophoretic variant of a human fibroblast protein with characteristics of smooth muscle tropomyosin

A charge variant of a protein (Tm: 3; molecular weight approximately 35,000) that co-migrates with human smooth muscle tropomyosin has been found in whole cell extracts from the fibroblasts of a father and his son. The variant protein co-purifies with Tm: 3, shifts with it to a higher apparent molecular weight on sodium dodecyl sulfate/polyacrylamide gel electrophoresis in the presence of 4 m-urea, is a component of the microfilaments, and has a peptide cleavage pattern identical to that of Tm: 3 and smooth muscle tropomyosin. The results indicate that Tm: 3 and the variant (Tm: 3.1) are smooth muscle tropomyosin, suggesting that normal human fibroblasts synthesize at least two tropomyosins (Tm: 3 and the non-muscle tropomyosin Tm: 4, molecular weight approximately 30,000) and that they are the products of separate genes.

Murine tissue amyloid protein AA. NH2-terminal sequence identity with only one of two serum amyloid protein (ApoSAA) gene products.

Amyloid protein AA is the presumptive fragment of an acute phase serum apolipoprotein, apoSAA. Two major murine apoSAA isotypes (apoSAA1 and apoSAA2) have been identified. The NH2-terminal amino acid sequences of purified murine apoSAA1 and apoSAA2 have been examined and compared with that of murine amyloid protein AA. Our results indicate that apoSAA1 and apoSAA2 are separate gene products and that amyloid protein AA has NH2-terminal amino acid sequence identity with only one of these isotypes, namely apoSAA2.

Structure and reactivity of cysteine residues in mitochondrial serine hydroxymethyltransferase.

Six cysteine-containing tryptic peptides were isolated and sequenced from rabbit liver mitochondrial serine hydroxymethyltransferase. 2 of the 6 cysteine residues were located on the surface of the enzyme. These 2 cysteine residues are sufficiently close to each other that they form a disulfide bond when oxidized by periodate. Another of the cysteine residues is exposed upon removal of the active site pyridoxal phosphate. The remaining three cysteines are buried and react with sulfhydryl reagents only when the enzyme is denatured. Blocking of one of the surface sulfhydryls with any of several sulfhydryl reagents results in increased catalytic activity when allothreonine is the substrate but decreased activity when serine and tetrahydrofolate are the substrates. The activation and inhibition effects are on Vmax and not on the affinity of the enzyme for its substrates. Of the six cysteine peptides from the mitochondrial enzyme, three show substantial homology with cysteine-containing peptides from the cytosolic form of the enzyme. For both enzyme forms, one of these homologous pairs is a cysteine residue on the surface of the enzyme. These results suggest that the mitochondrial and cytosolic forms of rabbit liver serine hydroxymethyltransferases are the products of separate genes.

Comparative studies of mouse liver cathepsin B and an analogous tumor thiol proteinase.

Cathepsin B (EC 3.4.22.1) and an analogous thiol proteinase were isolated from mouse liver and from a transplantable tumor induced by methylcholanthrene, respectively, by a sequence of steps involving salt fractionation and ion exchange and gel permeation chromatography. Both enzymes are capable of hydrolyzing N-benzyloxycarbonyl-L-Ala-L-Arg-L-Arg-4-methoxy-2-naphthylamide but are weakly active towards N-benzoyl-DL-arginine-2-naphthylamide. The specific activity of the liver enzyme towards these substrates is approximately 14 times greater than that of the tumor enzyme. Both enzymes show a single band with slight difference in mobility when subjected to gel electrophoresis at pH 4.5, but both exhibit a multiple banding pattern when examined by isoelectric focusing. The tumor enzyme has a somewhat higher molecular weight than the liver enzyme (33,000 versus 30,000) and possesses a slightly higher helical content (48% versus 40%) based on CD spectra. Both enzymes display maximum activity in the pH range of 5.5 to 7.0 and are irreversibly denatured above pH 7 and below pH 4. Both enzymes cross-react with antiserum towards the tumor enzyme. The liver enzyme displays a higher catalytic efficiency towards a series of oligopeptide substrates than the tumor enzyme, but is only one-third as active towards N-benzoyl-L-arginine-2-naphthylamide. Both proteinases exhibit similar patterns of inhibition by iodoacetate, chloroquine, leupeptin, antipain, and several peptide chloromethylketones. Despite what appear to be subtle differences in physical properties, amino acid composition data and peptide mapping revealed significant differences between these two enzymes reflective of extensive regions of non-identity. These results suggest that the tumor thiol protease and liver cathepsin B are products of separate genes and that the tumor enzyme is not likely an immediate precursor of the liver enzyme produced by post-translational modification.

Phosphorylation, maturational processing, and relatedness of strain colburn matrix proteins

The intracellular 66,000 D (66K) and 69,000 D (69K) matrix-like phosphoproteins of strain Colburn cytomegalovirus (CMV) have been compared with each other and with their electrophoretic counterparts in the virion. Three lines of evidence indicate that the 66K and 69K proteins are products of separate genes, and that the intracellular and virion :species are closely related. First, “pulse-chase” radiolabeling experiments showed that these proteins have separate precursors; that modification of each to the mature form correlated with phosphorylation; and that phosphorylation of the 69K precursor occurred more slowly than that of the 66K precursor, and resulted in a more dramatic slowing; of its electrophoretic mobility. Second, comparisons of the 66K and 69K proteins based on partial proteolysis of [ 35S]methionine-labeled proteins, using VS protease, and complete proteolysis of [ 32P]orthophosphate-labeled proteins, using trypsin or Pronase, provided no evidence of sequence relatedness. These analyses also suggested that the distribution of phosphorylated residues differs in the two proteins—clustered for the 69K and more disperse for the 66K. Phosphoamino acid analyses showed only phosphoserine in the 66K protein. The 69K protein contained, in addition to phosphoserine, an electrophoretically faster moving, unidentified spot. And third, immunological comparisons showed these proteins to exhibit little or no antigenic cross-reactivity. They did, however, demonstrate that the nuclear proteins are immunologically cross-reactive with their respective virion counterparts. Additional comparisons of these nuclear and virion proteins established that the virion 69K protein (v69) differs in electrophoretic mobility and net charge from the nuclear 69K protein but that it, as well as the virion 66K protein, has a two-dimensional phosphopeptide pattern similar to its nuclear counterpart.

Direct demonstration of glucocorticoid receptor phosphorylation by intact L-cells.

Glucocorticoid-sensitive L-cells were cultured for 18 h in the presence of [32P]orthophosphate and steroid-binding proteins of cytosol were separated by affinity chromatography and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing. Cytosol contains a major phosphoprotein of Mr = 92,000 and a minor phosphoprotein of Mr = 100,000, both of which bind glucocorticoids in a stereospecific, high affinity manner and have the same Mr as glucocorticoid receptor species that have been covalently labeled with the site-specific affinity ligand [3H] 9 alpha-fluoro-16-methyl-11 beta,17 alpha,21-trihydroxypregna-1, 4-diene-3,20-dione 21-mesylate. Cytosol from 32P-labeled, glucocorticoid-resistant L-cells possessing 5% of the steroid-binding capacity of sensitive cells contains very little of the Mr = 92,000 phosphoprotein and none of the Mr = 100,000 phosphoprotein. These observations provide strong evidence that the glucocorticoid receptor is phosphorylated by intact L-cells. The Mr = 92,000 protein is phosphorylated on serine and it can be resolved into two species using isoelectric focusing, consistent with the proposal that there is more than 1 phosphorylated serine/steroid-binding unit. The glucocorticoid-resistant L-cell line produces a unique phosphoprotein of Mr = 104,000 that is recovered in variable amounts after affinity chromatography. It is not known whether this phosphoprotein is a separate gene product or whether it represents a precursor with weak steroid-binding activity that is not cleaved in the resistant cell to the high affinity, Mr = 92,000 mature receptor form.

Evidence that the Ya and Yc subunits of glutathione transferase B (ligandin) are the products of separate genes.

A study of the structure of glutathione transferase B (ligandin) has been made with a view to understanding the relationship between the structures of the subunits of which it is composed. It consists of a mixture of a homodimer (YaYa) and a heterodimer (YaYc) in which the monomers are defined by their apparent molecular weights, that of Ya being 22000 and Yc 25000. Soluble tryptic peptides from the native homodimer YaYa have been compared with those from an artificial homodimer YcYc produced by rehybridization of native YaYc. Approximately 10 peptides specific to YaYa, 12 specific to YcYc and 21 common to both have been detected. Some of the above peptides are derived from variants of the monomers themselves. YaYa and YcYc have two C termini which are the same in both dimers, namely phenylalanine and lysine. Also there are four cysteinyl peptides, of which three are common to YaYa and YcYc and one specific to each. These results suggest that Ya and Yc are derived from at least two different but related genes.

Phenobarbital-induced rat liver cytochrome P-450. Purification and characterization of two closely related isozymic forms.

The "major" phenobarbital (PB)-induced cytochrome P-450 species present in livers of male Sprague-Dawley rats was resolved into two catalytically active heme-protein fractions on diethylaminoethyl cellulose. The two species, P-450 PB-4 (Mr = 49,000) and P-450 PB-5 (Mr = 51,000), were purified to homogeneity, and their chromatographic, spectral, catalytic, and structural properties were compared. P-450 BP-5 eluted earlier on hydroxylapatite and exhibited a more significant cholate-induced Type I spectral shift than P-450 BP-4. Very similar substrate specificity profiles were evident when the two isozymes were reconstituted with lipid, cytochrome P-450 reductase, and cytochrome b5 for oxidative metabolism of several xenobiotics, although P-450 PB-4 exhibited a higher specific catalytic activity (greater than or equal to 5-fold) with all substrates tested. Marked differences were also observed in the sensitivities of both isozymes to several P-450 inhibitors. In addition, P-450 PB-4 was greater than or equal to 10-fold more susceptible than P-450 PB-5 to suicide inactivation by two allyl-containing compounds, allylisopropylacetamide and secobarbital, providing a possible explanation of the previously observed partial inactivation by such compounds of phenobarbital-induced P-450 activity in liver microsomes. One-dimensional peptide maps of the two isoenzymes were highly similar. Antibody raised against purified Long Evans rat liver P-450b (Thomas, P. E., Korzeniowski, D., Ryan, D., and Levin, W. (1979) Arch. Biochem. Biophys. 192, 524-532) cross-reacted with P-450 PB-4 and P-450 PB-5. NH2-terminal sequence analysis demonstrated that the first 31 residues of both PB-4 and PB-5 were identical. These sequences indicated that a highly hydrophobic terminal segment, observed previously for other P-450s as well, is followed by a cluster of basic residues, suggesting that the NH2-terminal portion of these P-450s might be involved in membrane anchoring. Although it is unclear whether P-450 PB-4 and P-450 PB-5 are separate gene products or are related by post-translational modifications, this present demonstration of closely related isozymic forms suggests the possible added complexity of microheterogeneity for this family of microsomal monooxygenases.

Receptor properties of corticosteroid binder IB

Corticosteroid binder IB, present in liver and kidney, is pronounced in liver cytosol after injection of [ 3H]triamcinolone acetonide. Following injection of the radioactive ligand, livers homogenized in the presence of 20 m m molybdate, 2 m m leupeptin hemisulfate, 2 m m antipain, or 2 m m phenylmethylsufonyl fluoride produce cytosols with Chromatographie profiles of binders II and IB identical to controls, as determined by DEAE-Sephadex chromatography, suggesting that IB is a cellular constituent rather than a product of protease action (sensitive to the above inhibitors) after cell breakage. Generation of IB in kidney cytosols in vitro appears to be unrelated to protease activity. Liver binder IB has an S value of 5–6 and a Stokes radius of about 26 Å producing a calculated range of molecular weight from 40,000 to 50,000 with frictional coefficient and axial ratio close to spherical values. As expected of a steroid receptor, IB, like II, binds to DNA and to liver cell nuclei but IB binds more tightly as evidenced by the fact that KCl is more effective in eluting II than IB from nuclei. Because recovery of bound radioactivity from acceptors is sometimes difficult to achieve, indirect experiments have been used frequently to determine the binding. Pyridoxal phosphate extracts liver IB and II equally from nuclei but spermidine is ineffective. While IB and II can be extracted partially from nuclei by pancreatic DNase I, more binder II is extracted by this method than IB. Micrococcal nuclease is poorly effective in either case. Binder II is extracted to a greater degree from DNA-cellulose than is IB by spermidine, MgCl 2, pyridoxal phosphate, and NaCl. IB binds more extensively to homodeoxypolymers than II. The extent of binding of liver IB to homodeoxypolymers is in the order: poly(dC) ≥ poly(dG) > poly(dA) ⪢ poly(dT), whereas the order for liver binder II is: poly(dG) ≥ poly(dT) > poly(dC) ⪢ poly(dA). Binders IB and II may be separate gene products or IB may arise in the cell from post-translational action. In the latter case, the activity of a protease cannot be ruled out.

Isolation and Structural Characterization of the Polypeptide Subunits of Membrane Glycoprotein IIb-IIIa From Human Platelets

Abstract We have previously demonstrated the isolation of platelet membrane glycoprotein IIb-IIIa by affinity chromatography with a specific monoclonal antibody. We have now separated the polypeptide subunits IIb and IIIa of the isolated glycoprotein by preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis and have compared their structural features. Both IIb and IIIa contain approximately 15% carbohydrate, but IIIa contains a larger percentage of mannose residues, suggesting the presence of high mannose as well as complex N- linked oligosaccharide chains. The amino acid compositions are sufficiently similar to imply areas of sequence homology between the two subunits. To examine further the relationship between the subunits, we digested a mixture of 125I-IIb and 131I-IIIa with trypsin and then separated the radiolabeled peptides by high performance liquid chromatography. The resultant peptide maps of IIb and IIIa are completely different. This indicates that neither subunit is derived from the other and suggests that polypeptides IIb and IIIa are products of separate genes.

Isolation and structural characterization of the polypeptide subunits of membrane glycoprotein IIb-IIIa from human platelets

We have previously demonstrated the isolation of platelet membrane glycoprotein IIb-IIIa by affinity chromatography with a specific monoclonal antibody. We have now separated the polypeptide subunits IIb and IIIa of the isolated glycoprotein by preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis and have compared their structural features. Both IIb and IIIa contain approximately 15% carbohydrate, but IIIa contains a larger percentage of mannose residues, suggesting the presence of high mannose as well as complex N-linked oligosaccharide chains. The amino acid compositions are sufficiently similar to imply areas of sequence homology between the two subunits. To examine further the relationship between the subunits, we digested a mixture of 125I-IIb and 131I-IIIa with trypsin and then separated the radiolabeled peptides by high performance liquid chromatography. The resultant peptide maps of IIb and IIIa are completely different. This indicates that neither subunit is derived from the other and suggests that polypeptides IIb and IIIa are products of separate genes.

41] Cell-free translation of elastin mRNAs

To cover fully the system for effective cell-free translations of elastin mRNAs, the methodology is divided into four sections dealing with (1) isolation of RNA and preparation of mRNA, (2) preparation of the mRNA-dependent reticulocyte lysate and optimal conditions for the cell-free translation of elastin mRNAs, (3) identification of tropoelastins in a cell-free translation assay, and (4) quantitation of tropoelastins in the cell-free translation system. It is important to note that the discussion of the cell-free synthesis of elastin involves the synthesis of two proteins designated tropoelastins a and b. All the evidence to date suggests strongly that these two tropoelastins are separate gene products. Consequently, the methodologies described are geared not only for the production of soluble elastin, but also for the separate identification of tropoelastins a and b.

Characterization of viral polyproteins in cells transformed and producing Moloney murine sarcoma virus-124

The viral proteins of Moloney murine sarcoma virus-transformed mouse cells (G8–124), that overproduce the sarcoma virus relative to helper leukemia virus, were examined by immunoprecipitation, sizing by gel electrophoresis and peptide mapping. Using antisera to the viral core proteins, a p10-deficient core polyprotein of 63 000 daltons (P63 gag) was detected which was found to be a product of the MuSV-124 genome. Helper virus proteins Pr67 gag, gPr85 gag, Pr200 gag-pol, and gPr83 env were also detected and characterized. An unusual helper virus precursor polyprotein of 93 000 daltons was also detected and characterized. It was labeled with [ 3H]fucose, suggesting that it was an intermediate precursor containing gp70 on which further modification of the core oligosaccharide had occurred relative to gPr83 env. The usual viral proteins were also found in sarcoma virus particles and they were undistinguishable in size from those of Moloney murine leukemia virus (cl-1 strain). These experiments together with pulse-labeling experiments performed in the presence of the arginine analog canavanine, which prevents proteolytic cleavage, suggest that the product derived from the Mo-MuSV-specific sequences must be expressed as a separate gene product since we were unable to detect in transformed cells a polyprotein containing both viral (e.g., ‘ gag’, ‘ pol’ or ‘ env’) and non-viral components. Cell-free protein synthesis studies performed with Mo-MuSV-124 genomic RNA have confirmed this interpretation and have identified three size classes of polypeptides as translation products of the sarcoma virus genome. They are P63 gag, P42-P38 and P23. Intracellular p63 gag and cell-free synthesized P63 gag appear to have identical sizes, antigenic determinants and tryptic peptides. The P42-P38 proteins contain core protein determinants. P23 is not related to the products of the replication genes of MuLV. Thus, P23 is a candidate for the ‘ src’ gene product of Mo-MuSV-124.

Avian feather keratins: Molecular aspects of structural heterogeneity

AbstractSolubilized SCM‐Keratins from feathers of a tern (Sterna hirundo) and turkey (Meleagris gallopavo) were fractionated by gel‐filtration and ion‐exchange chromatography. Tryptic peptide maps were prepared and amino‐acid compositions determined. Subsequent comparison indicated that within each species the FKM were closely related, but separate gene products. Interspecific comparison showed species specificity. The data were compared with the homologous FKM of other species and to amino‐acid composition data of the unfractionated SCM‐KM from other tissues.These results support the proposition that feather keratins represent a family of closely related gene products. The primary function of the multiplicity of structural elements is in the macro‐molecular organization of the tissue. The monomers can self‐associate to form both the filamentous structures and an intermolecular matrix. The FKM also provide tissue specific organizational information. The chemical composition and distribution of the different FKM presumably determines the unique morphological and functional aspects of the feathers.

Multiple actins in Drosophila melanogaster.

The tissue and developmental specificities of the three Drosophila isoactins, originally identified in primary myogenic cultures and in the permanent Schneider L-2 cell line, have been investigated. Of these three isoactins (I, II, and III), actins I and II are stable and actin III is unstable. Two-dimensional polyacrylamide gel electrophoretic analyses of total cellular extracts after 1-h [(35)S]methionine pulses were performed on a large variety of embryonic, larval, and adult muscle and nonmuscle tissues. The results suggest that isoactins II and III are generalized cellular actins found in all drosophila cell types. Actin I, on the other hand, is muscle-associated and is found exclusively in supercontractile muscle (such as larval body wall and larval and adult viscera) including primary myogenic cell cultures. Although actin I synthesis is not detectable during very early embryogenesis, it is detectable by 25 h and actin I is a major stable actin in all larval muscle tissues. Actin I is synthesized in reduced amounts relative to the other actins in late third instar larvae but is again a major product of actin synthesis in the adult abdomen. A stable actin species with the same pI as actin III has been identified in the adult thorax and appears to be unique to flight muscle tissue. This new stable form of thoracic actin may be the result of a stabilization of the actin III found in other tissues or may be an entirely separate gene product.