Methionine-containing Tryptic Peptides Research Articles

Selective solid-phase isolation of methionine-containing peptides and subsequent matrix-assisted laser desorption/ionisation mass spectrometric detection of methionine- and of methionine-sulfoxide-containing peptides.

Methionine residues and the oxidised forms in proteins are becoming more and more important in view of their biological function. In particular, methionine sulfoxide seems to have a regulatory function. This paper presents a fast strategy for simultaneous determination of methionine- and methionine-sulfoxide-containing peptides, involving application of methionine-specific solid-phase reagent chemistry combined with matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS). In the first step, methionine-containing peptides are covalently bound as sulfonium salts to glass beads, whereas methionine-sulfoxide-containing peptides and other methionine-free peptides are not bound and are washed out. The wash solution is used for MALDI-MS analysis to determine the molecular masses of these peptides and to perform, if necessary, seamless post-source decay (PSD) fragment ion analysis. Methionine-sulfoxide-containing peptides can be identified due to the characteristic metastable loss of methanesulfenic acid from the protonated molecules. In the second step, the bound peptides are cleaved from the matrix of the beads by addition of 2-mercaptoethanol at pH 8.5-8.8. The resulting peptides, mainly methionine-containing peptides, are analysed in a straightforward manner by MALDI-MS and seamless PSD. The strategy allows the fast identification of methionine- and methionine-sulfoxide-containing peptides even in complex tryptic digests, as demonstrated here for the glycoprotein antithrombin. These results show that sometimes methionine-containing tryptic peptides are not detected due to steric restrictions (e.g. glycosylation near the methionine residue) on the binding reaction, and that, on the other hand, some methionine-free peptides can be quite strongly bound non-covalently to the matrix of the beads. The latter observation indicates the necessity of seamless PSD fragment ion analysis for unambiguous identification. Furthermore, there are indications that oxidation of some methionine residues occurred to a minor extent during the solid-phase isolation steps.

Isolation and Isotope Labeling of Cysteine- and Methionine-containing Tryptic Peptides: Application to the Study of Cell Surface Proteolysis

Inexpensive methods were developed for isolating and isotopically labeling tryptic peptides that contain either cysteine or methionine. After covalently capturing cysteine-containing peptides with pyridyl disulfide reactive groups on agarose beads, extensive wash steps were applied, and the attached peptides were released using a reducing agent. This approach results in less nonspecifically bound peptides and eliminates the possibility of generating avidin peptide background ions that can arise when using methods based on biotin and avidin (e.g. isotope-coded affinity tag). The thiols were alkylated using either N-ethyl- or N-D5-ethyl-iodoacetamide, both of which can be synthesized in a single step using inexpensive reagents. This isotopic labeling does not greatly increase the peptide mass, nor does it affect the peptide ion charge state in electrospray ionization. In addition, methionine-containing peptides were captured using commercially available methionine-reactive beads, and relative quantitation of peptides was achieved by isotopic labeling of amino groups using activated esters of either nicotinic acid or D4-nicotinic acid. These methods were used to study the metalloprotease-mediated shedding of cell surface proteins from a mouse monocyte cell line that had been treated with a phorbol ester and lipopolysaccharide. In addition to the identification of proteins previously determined to be inducibly shed, three new shed proteins were identified: CD18, ICOS ligand, and tumor endothelial marker 7-related protein.

Reagent or myeloperoxidase-generated hypochlorite affects discrete regions in lipid-free and lipid-associated human apolipoprotein A-I

We have previously shown that the modification of high-density lipoprotein subclass 3 (HDL(3)) by HOCl transformed an anti-atherogenic lipoprotein into a high-uptake form for macrophages and caused a significant impairment of cholesterol efflux capacity [Panzenboeck, Raitmayer, Reicher, Lindner, Glatter, Malle and Sattler (1997) J. Biol. Chem. 272, 29711-29720]. To elucidate the consequences of treatment with OCl(-) on distinct regions in apolipoprotein A-I (apo A-I), lipid-free and lipid-associated apo A-I were modified with increasing molar ratios of NaOCl or HOCl generated by the myeloperoxidase/H(2)O(2)/Cl(-) system. CD analysis revealed a pronounced decrease in alpha-helicity for lipid-free apo A-I modified by NaOCl, whereas lipid-associated apo A-I was less affected. The modification of apo A-I by NaOCl (molar oxidant-to-lipoprotein ratio 6:1) resulted in the formation of two distinct oxidized forms of apo A-I with molecular masses 32 or 48 atomic mass units (a.m.u.) higher than that of native apo A-I, indicating the addition of two or three oxygen atoms to the native protein. HPLC analysis of tryptic digests obtained from lipid-free and lipid-associated apo A-I modified with increasing oxidant-to-apolipoprotein molar ratios revealed a concentration-dependent modification of apo A-I: at a low molar oxidant-to-lipoprotein ratio (5:1) the peaks corresponding to the methionine-containing tryptic peptides T11 (residues 84-88), T16 (residues 108-116) and T22 (residues 141-149), located in the central region of apo A-I, disappeared. Their loss was accompanied by the formation of three oxidation products with a molecular mass 16 a.m.u. higher than that of the native peptides. This indicates the addition of oxygen, most probably caused by the oxidation of Met(86), Met(112) and Met(148) to the corresponding methionine sulphoxides. At a molar NaOCl-to-apo A-I ratio of 10:1 the disappearance of peptides T1 (residues 1-10), T7 (residues 46-59) and T9 (residues 62-77) was accompanied by the occurrence of new peaks 33.5 and 33.1 a.m.u. higher than those of the native peptides. Amino acid analyses of peptides T7 and T9 after modification with NaOCl confirmed that Phe(57) and Phe(71) were primary targets for oxidation by HOCl. GLC-MS analysis of hydrolysates obtained from OCl(-)-modified T7, T9, apo A-I and HDL(3) confirmed that Phe residues are an early target for OCl(-) modification. At molar NaOCl-to-apo A-I ratios of 25:1, the peak areas of peptides T31 (residues 189-195) and T32 (residues 196-206) decreased markedly. Most importantly, incubation of apo A-I with the myeloperoxidase/H(2)O(2)/Cl(-) system (the source of HOCl in vivo) resulted in almost identical modification patterns to those observed with reagent NaOCl.

Nucleoprotein phosphorylated on both serine and threonine is preferentially assembled into the nucleocapsids of measles virus

The nucleoprotein (N) in the nucleocapsids of measles virus (MV) has different conformation and antigenicity than the free N-protein in MV-infected cells. These two forms of N-protein have identical methionine-containing tryptic peptides. The free N-protein contains 4 phosphorylated tryptic peptides. However, the nucleocapsid-associated N-protein has an additional phosphorylated peptide not found in the free N-protein. The free N-protein is phosphorylated only on serine residues, whereas the nucleocapsid-associated N-protein is phosphyralated on both serine and threonine residues. The MV N-protein expressed from a cloned gene in primate cells is also phosphorylated on both serine and threonine residues. These results suggest that cellular kinases phosphorylate the MV N-protein, and N-protein with phosphorylated serine and threonine is preferentially assembled into the viral nucleocapsids.

Identification of methionine-containing tryptic peptides of unstable β-tubulin separated by reverse-phase high-performance liquid chromatography

Methods for examining altered regions in unstable mutant proteins are described. The strategy is illustrated using assembly defective Chinese hamster β-tubulin subunits that are rapidly degraded in the cell. These unstable proteins are metabolically labeled to high specific activity and isolated as spots on two-dimensional gels. Conditions for the generation of tryptic peptides from gel pieces containing β-tubulin and their subsequent resolution by HPLC have been worked out. Through a combination of dual labeling with various tritiated amino acids and [ 35S]methionine as well as partial sequence analysis, the identification of several HPLC peaks with the known sequence of β-tubulin has been accomplished. This technique should greatly aid attempts to map the sites of mutational alterations in β-tubulin polypeptides, and the general strategy should be readily applicable to other mutant proteins.

Identification of methionine-110 as the residue covalently modified in the electrophilic inactivation of D-amino-acid oxidase by O-(2,4-dinitrophenyl) hydroxylamine.

The reaction of O-(2,4-dinitrophenyl)hydroxylamine with D-amino-acid oxidase leads to complete inactivation which can be protected against by the competitive inhibitor benzoate [D'Silva, C., Williams, C. H., Jr., & Massey, V. (1986) Biochemistry 25, 5602-5608]. The residue modified has been identified as methionine-110. Differential high-performance liquid chromatography mapping of tryptic digests of D-amino-acid oxidase modified in the absence and presence of benzoate allows the isolation of a single methionine-containing tryptic peptide corresponding to residues 100-115 and referred to as T6-T7. In unmodified enzyme, the bond involving Arg-108 is readily cleaved and T6 and T7 are isolated. Brief treatment of peptide T6-T7 with carboxypeptidase Y released residues 112-115, and the residual peptide was isolated in good yield. Further treatment of this peptide (residues 100-111) with carboxypeptidase Y released Val and an unknown amino acid that comigrated with synthetically prepared S-aminomethionine sulfonium salt. The unknown compound and S-aminomethionine break down to methionine on treatment with dithiothreitol.

Absence of a structural basis for intracellular recognition and differential localization of nuclear and plasma membrane-associated forms of simian virus 40 large tumor antigen.

The simian virus 40 large tumor antigen (T-ag) is found in both the nuclei (nT-ag) and plasma membranes (mT-ag) of simian virus 40-infected or -transformed cells. It is not known how newly synthesized T-ag molecules are recognized, sorted, and transported to their ultimate subcellular destinations. One possibility is that these events depend upon structural differences between nT-ag and mT-ag. To test this possibility, we compared the structures of nT-ag and mT-ag from simian virus 40-infected cells. No differences between the two forms of T-ag were detected by migration in polyacrylamide gels, by Staphylococcus aureus V8 partial proteolytic mapping of methionine- or proline-containing peptides, or by two-dimensional tryptic peptide mapping of methionine-containing peptides. The carboxy-terminal, methionine-containing tryptic peptide was identified in the two-dimensional maps and was shown to be identical in nT-ag and mT-ag. Thus, a structural basis for the recognition and differential localization of T-ags could not be demonstrated. The carboxy terminus of the T-ag encoded by mutant dlA2413 is derived from the alternate open reading frame of the simian virus 40 early region, in analogy with the theoretical early gene product, T*-ag. We used this mutant to identify peptides unique to T*-ag. None of these peptides were detected in maps of mT-ag; only wild-type T-ag-specific peptides were found. These findings suggest that T*-ag does not represent the membrane-associated form of T-ag, but that mT-ag is encoded within the same reading frame used for nT-ag.

Absence of a structural basis for intracellular recognition and differential localization of nuclear and plasma membrane-associated forms of simian virus 40 large tumor antigen.

The simian virus 40 large tumor antigen (T-ag) is found in both the nuclei (nT-ag) and plasma membranes (mT-ag) of simian virus 40-infected or -transformed cells. It is not known how newly synthesized T-ag molecules are recognized, sorted, and transported to their ultimate subcellular destinations. One possibility is that these events depend upon structural differences between nT-ag and mT-ag. To test this possibility, we compared the structures of nT-ag and mT-ag from simian virus 40-infected cells. No differences between the two forms of T-ag were detected by migration in polyacrylamide gels, by Staphylococcus aureus V8 partial proteolytic mapping of methionine- or proline-containing peptides, or by two-dimensional tryptic peptide mapping of methionine-containing peptides. The carboxy-terminal, methionine-containing tryptic peptide was identified in the two-dimensional maps and was shown to be identical in nT-ag and mT-ag. Thus, a structural basis for the recognition and differential localization of T-ags could not be demonstrated. The carboxy terminus of the T-ag encoded by mutant dlA2413 is derived from the alternate open reading frame of the simian virus 40 early region, in analogy with the theoretical early gene product, T*-ag. We used this mutant to identify peptides unique to T*-ag. None of these peptides were detected in maps of mT-ag; only wild-type T-ag-specific peptides were found. These findings suggest that T*-ag does not represent the membrane-associated form of T-ag, but that mT-ag is encoded within the same reading frame used for nT-ag.

Identification and preferential expression in thymic and bursal lymphocytes of a c-ets oncogene-encoded Mr 54,000 cytoplasmic protein.

The avian retrovirus E26 is unique among acute leukemia viruses in its ability to induce transformation of cells belonging to either the myeloid or erythroid lineage. The genome of E26 carries two oncogenes, v-myb and v-ets, that are derived from distinct cellular loci, c-myb and c-ets. We have constructed a plasmid vector that allows expression of part of the coding region of v-ets in a bacterial host. Antisera to the bacterially synthesized ets protein specifically precipitated the E26-encoded P135gag-myb-ets transforming protein. These antisera permitted us to identify a chicken c-ets-encoded protein of Mr 54,000 (P54c-ets) that shares 7 out of 10 of its major [35S]methionine-containing tryptic peptides with the v-ets-encoded domain of P135gag-myb-ets. Unlike P135gag-myb-ets and the Mr 75,000 translation product of c-myb (P75c-myb), which are nuclear proteins, P54c-ets was found to be predominantly cytoplasmic. P54c-ets is expressed at low levels in most cell lines and tissues tested, including bone marrow cells and circulating lymphocytes. P54c-ets, together with a minor but closely related Mr 56,000 protein, was found to be expressed at high levels in chicken thymocytes and bursal lymphocytes.

Immunological and Biochemical Characterization of HZ2 Feline Sarcoma Virus and Abelson Murine Leukaemia Virus Translation Products

The extent of homology between the translation products of the HZ2 strain of feline sarcoma virus (HZ2-FeSV) and the Abelson murine leukaemia virus (A-MuLV) was examined immunologically and biochemically. Antiserum prepared against the v-abl-encoded determinants of the A-MuLV polyprotein P120gag-abl was also found to precipitate specifically the 98K mol. wt. HZ2-FeSV protein (P98gag-abl). The basis for this immunological crossreactivity was indicated by the findings that the two proteins had at least six [35S]methionine-containing tryptic peptides and at least eight [35S]methionine-containing chymotryptic peptides in common. Each of the two proteins also had tryptic and chymotryptic peptides which were unique. Both proteins were associated with tyrosyl kinase activities which exhibited some similar biochemical properties in vitro. However, the HZ2-FeSV-associated activity was much more sensitive to competitive inhibition by nucleoside and deoxynucleoside diphosphates than was the A-MuLV-associated activity. These results suggest that, while the gag-abl translation products of these two independent isolates of transforming retrovirus are highly related structurally and functionally, the differences in structure contribute to differences in enzyme activity. Further comparative studies of these two proteins should play an important role in determining their roles in induction of two different types of malignancy: lymphosarcoma in the case of the A-MuLV protein and fibrosarcoma in the case of the HZ2-FeSV protein.

Intracisternal A-particle gene expression in normal mouse thymus tissue: gene products and strain-related variability.

Intracisternal A-particle (IAP)-specific sequences were 5- to 10-fold enriched in polyadenylated RNA from BALB/cJ thymus as compared with RNAs from liver, spleen, and kidney. The major transcripts of 7.2 and 5.4 kilobases were the same size as those found in an IAP-rich neuroblastoma cell line. The absolute levels and proportions of these transcripts varied in thymuses from mice of different inbred strains. With antiserum prepared against p73, the main IAP structural protein, several size classes of IAP-related proteins were immunoprecipitated from extracts of thymus cells incubated with [35S]methionine; these included p73 itself and a group of polypeptides in the size range of 114 to 120 kilodaltons (p114-p120). The inbred strains showed marked characteristic differences in the electrophoretic patterns of their IAP-related proteins. Earlier studies showed that the 7.2-kilobase RNA from neuroblastoma IAPs coded for p73 in a cell-free translation system. Correlations between the RNA and protein patterns in thymuses of the different inbred strains indicated that 5.4-kilobase RNA gives rise to the p114-p120 polypeptides. Metabolically labeled p120 was found to include methionine-containing tryptic peptides of p73 plus additional peptides consistent with its larger size. In vivo labeling kinetics showed that the p114-p120 polypeptides were not major precursors of p73 in intact neuroblastoma cells. This study shows that IAP gene expression in mouse thymus is genetically determined and that a novel class of IAP-related polypeptides can be expressed independently of the major particle structural protein.

Intracisternal A-Particle Gene Expression in Normal Mouse Thymus Tissue: Gene Products and Strain-Related Variability

Intracisternal A-particle (IAP)-specific sequences were 5- to 10-fold enriched in polyadenylated RNA from BALB/cJ thymus as compared with RNAs from liver, spleen, and kidney. The major transcripts of 7.2 and 5.4 kilobases were the same size as those found in an IAP-rich neuroblastoma cell line. The absolute levels and proportions of these transcripts varied in thymuses from mice of different inbred strains. With antiserum prepared against p73, the main IAP structural protein, several size classes of IAP-related proteins were immunoprecipitated from extracts of thymus cells incubated with [35S]methionine; these included p73 itself and a group of polypeptides in the size range of 114 to 120 kilodaltons (p114-p120). The inbred strains showed marked characteristic differences in the electrophoretic patterns of their IAP-related proteins. Earlier studies showed that the 7.2-kilobase RNA from neuroblastoma IAPs coded for p73 in a cell-free translation system. Correlations between the RNA and protein patterns in thymuses of the different inbred strains indicated that 5.4-kilobase RNA gives rise to the p114-p120 polypeptides. Metabolically labeled p120 was found to include methionine-containing tryptic peptides of p73 plus additional peptides consistent with its larger size. In vivo labeling kinetics showed that the p114-p120 polypeptides were not major precursors of p73 in intact neuroblastoma cells. This study shows that IAP gene expression in mouse thymus is genetically determined and that a novel class of IAP-related polypeptides can be expressed independently of the major particle structural protein.

Studies on the primary structure of .ALPHA.-amylase from Bacillus subtilis var. amylosacchariticus. Part IV. Alignments of tryptic peptides in CNBr fragments of .ALPHA.-amylase from Bacillus subtilis var. amylosacchariticus.

To determine the primary structure of the α-amylase produced by Bacillus subtilis var. amylosacchariticus, we have reported the isolation of thirty-four tryptic peptides and eight CNBr fragments from the enzyme. Since the alignment of the eight CNBr fragments was made by matching with six methionine-containing tryptic peptides, the order of tryptic peptides within each CNBr fragment was determined. In the case of four small CNBr fragments, sequence analyses using an automated sequence analyzer established the peptide orders within these fragments. For larger fragments, further fragmentation was done using chymotrypsin or staphylococcal protease V8 and the resultant peptides were isolated and sequenced. Consequently, the peptide orders within three out of four large CNBr fragments were established.

Studies on the primary structure of .ALPHA.-amylase from Bacillus subtilis var. amylosacchariticus. Part III. Isolation and alignment of CNBr fragments of .ALPHA.-amylase from Bacillus subtilis var. amylosacchariticus.

We examined the primary structure of α-amylase produced by Bacillus subtilis var. amylosacchariticus by isolation and characterization of CNBr fragments of the enzyme. By solubilization and precipitation in a buffer, the fragments were first fractionated into two. The soluble fraction was fractionated by Bio-Gel P-30, and three fragments were obtained. The insoluble fraction was fractionated by SP-Sephadex C-25 and further purified by Bio-Gels, and five fragments were isolated. Amino acid sequences near the N- and C-terminus were determined with the eight CNBr fragments. By matching the sequences with those of methionine-containing tryptic peptides, alignment of the eight CNBr fragments was determined.

Isolation and characterization of the two giant secretory proteins in salivary gland of Chironomus tentans.

The two giant secretory proteins, sp-Ia and sp-Ib, in salivary-gland cells of the larva of the fly Chironomus tentans, were isolated by preparative gel electrophoresis and characterized chemically. Their amino acid compositions are dominated by polar amino acids, with about 30% of basic amino acid residues. Crossed immunoelectrophoresis of sp-Ia and sp-Ib provided evidence that they share antigenic determinants. They also have major methionine-containing tryptic peptides in common. CNBr cleavage of sp-Ib gives a small number of low-Mr fragments, indicating that this protein has a repetitive structure.

A 61,000-dalton truncated large T-antigen is uniformly expressed in hamster cells transformed by polyomavirus.

Various polyomavirus-transformed hamster cell lines derived from tumors or from infected hamster cell cultures synthesized polyoma middle and small tumor (T)-antigens but no full-size large T-antigen. Instead, all cell lines produced the same or similar polyoma T-antigen-related proteins of ca. 61 kilodaltons (kDal). Like large T-antigen synthesized in lytically infected mouse cells, the 61-kDal proteins were phosphoproteins showing electrophoretic and charge heterogeneities. Chromatographic analysis of the methionine-containing tryptic peptides indicated that the 61-kDal proteins were truncated forms of large T-antigen comprising amino acid residues 1 to 485 (+/- 25). Analysis of viral DNA present in hamster chromosomal DNA of three independently isolated cell lines confirmed that synthesis of the 61-kDal proteins was due to a discontinuity in the large T-antigen coding sequence, most likely located between 7 and 8.9 map units on the polyoma DNA map. The three cell lines yielded essentially the same patterns of viral DNA-containing restriction enzyme fragments, suggesting that insertion of viral DNA into the hamster chromosomes took place at closely similar sites.

Structural comparisons of wild-type and nuclear transport-defective simian virus 40 large tumor antigens

PARA(nT) is a defective SV40-adenovirus 7 hybrid virus which contains the entire early region of the SV40 genome and codes for the synthesis of SV40 large tumor antigen (T-ag). A transport-defective variant of this hybrid, PARA(cT), encodes T-ag that is not transported to the nucleus, but accumulates in the cytoplasm. The structures of T-ags extracted from wild-type (WT) SV40-, PARA(nT)-, and PARA(cT)-infected cells were compared by peptide mapping. All three types of T-ag underwent considerable degradation when extracted using Tris-buffered Nonidet P-40 at pH 8.0. The addition of 200 μM leupeptin to the extraction buffer significantly inhibited this degradation. Comparison of methionine-containing tryptic peptides revealed no differences among the T-ags, suggesting that their primary structures are similar or identical. Phosphopeptide mapping revealed no differences between SV40- and PARA(nT)-encoded T-ags. In contrast, PARA(cT)-encoded T-ag lacked a prominent phosphopeptide that was present in both of the others. The possible relevance of this difference in phosphorylation to the transport defect is discussed.

Histidine decarboxylase of Lactobacillus 30a. Sequences of the overlapping peptides, the complete alpha chain, and prohistidine decarboxylase.

The complete amino acid sequence of the alpha chain of histidine decarboxylase of Lactobacillus 30a has been established by isolation and analysis of the eight methionine-containing tryptic peptides of this chain. These peptides provide the overlaps required to order all nine peptides derived by complete cyanogen bromide cleavage of the alpha chain (Huynh, Q.K., Vaaler, G.L., Recsei, P.A., and Snell, E.E. (1984) J. Biol. Chem. 259, 2826-2832). Ordering of six of the latter peptides was confirmed by isolation and analysis of four peptides derived by incomplete cyanogen bromide cleavage. The alpha chain is composed of 226 residues and has a molecular weight of 24,892 calculated from the sequence. These results and the previously determined sequence of the beta chain (Vaaler, G.L., Recsei, P.A., Fox, J.L., and Snell, E.E. (1982) J. Biol. Chem. 257, 12770-12774) establish the complete amino acid sequence of the enzyme and of the pi chain of prohistidine decarboxylase. The latter is composed of 307 amino acids and has a calculated molecular weight of 33,731. Four segments of the pi chain sequence are repeated. The bond between Ser-81 and Ser-82 that is cleaved during proenzyme activation is in an uncharged portion of the sequence that is rich in serine and threonine residues and is predicted to be part of a beta sheet structure.

Genetic diversity in natural populations of mammalian reoviruses: tryptic peptide analysis of outer capsid polypeptides of murine, bovine, and human type 1 and 3 reovirus strains.

We have studied the structural relationships between the outer capsid polypeptides of eight murine, bovine, and human isolates of type 1 and 3 mammalian reoviruses. Our results show that the outer capsid polypeptides of reoviruses isolated from different mammalian species, in different years and different geographical areas, have both conserved and unique methionine-containing tryptic peptides. We found that tryptic peptides from mu 1C polypeptides of two human, one murine, and two bovine type 3 isolates and one human and two bovine type 1 reoviruses are highly conserved. Our data show that only one tryptic peptide pattern of the mu 1C polypeptide (encoded by the M2 gene) was present in reoviruses isolated from the three different mammalian species. The mu 1C polypeptide of the type 3 Dearing strain contained one tryptic peptide not found in any other reovirus isolate examined. In marked contrast to the mu 1C polypeptides, the sigma 3 polypeptides (encoded by the S4 gene) of three type 1 and three type 3 isolates were divided into two patterns based on significant differences in their tryptic peptides. In addition, at least seven tryptic peptides were conserved among the sigma 3 polypeptides of all virus strains examined. The sigma 3 polypeptide of the type 3 Dearing strain was distinguishable from the sigma 3 polypeptides of all other strains examined. The one mu 1C and two sigma 3 tryptic peptide patterns were found to occur interchangeably in isolates of type 1 or type 3. About 1/3 of the tyrosine-containing tryptic peptides of sigma 1 polypeptides of four type 3 isolates examined were conserved. Comparison of peptide differences in sigma 1 polypeptides of these isolates showed that each had one or more unique tryptic peptides, suggesting that the S1 genes coding for these polypeptides had undergone genetic drift or, alternatively, that there are at least two tryptic peptide patterns present among the sigma 1 polypeptides of these isolates. Our results suggest that genetic drift and reassortment are the most likely explanation for the extensive genetic diversity found in natural populations of mammalian reoviruses.

Isolation of a new feline sarcoma virus (HZ1-FeSV): Biochemical and immunological characterization of its translation product

A new strain of feline sarcoma virus, designated HZ1-FeSV, was isolated from a 4-year-old domestic cat with multicentric fibrosarcoma. A primary tumor cell line was established and virus produced from that line was found to induce foci in feline embryonic lung fibroblasts (FLF3) and mink lung fibroblasts (CCL64) in tissue culture and fibro-sarcomas in inoculated 10-week-old kittens. The derivation of transformed nonproducer clones of FLF3 and CCL64 cells containing helper virus-rescuable, focus-forming activity indicated that HZ1-FeSV was defective for replication. The only discernible translation product of the HZ1-FeSV genome in cultured cells was a 100,000-Da polyprotein (P100) which contained amino-terminal sequences of the FeLV gag gene precursor protein covalently linked to a sarcoma virus-specific domain. Immunoprecipitates containing P100 exhibited a protein kinase actvity capable of phosphorylating tyrosine residues of P100. Immunologically, P100 was highly cross-reactive with gag-fes polyproteins encoded by two previously characterized strains of FeSV, the GA- and the ST-FeSV. By comparison of methionine-containing tryptic peptides, the HZ1-FeSV protein was shown to be more closely related to the GA-FeSV protein than to the ST-FeSV protein, but to be distinguishable from both other proteins.