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E Appella

Publications and source records attributed to E Appella.

At least 307 records · Page 17Linked to original sources
PubMed1982

Complete sequence analysis of cDNA clones encoding rat whey phosphoprotein: homology to a protease inhibitor.

Lactoprotein clones have been isolated from a rat mammary gland recombinant library of cDNA plasmids. Clones p-Wp 52 and p-Wp 47 were shown by hybrid selection, in vitro translation, and immunoprecipitation to represent a cloned DNA sequence encoding rat whey phosphoprotein. We report here the nucleotide sequence of the cDNA insert of p-Wp 52 and shows that it encodes the complete whey phosphoprotein sequence. The encoded sequence shows a high content of half-cystine, glutamic acid, aspartic acid, and serine but an absence of tyrosine. The half-cystines appear in unique arrangements and are repeated in two domains of the protein. The second domain has striking similarities with the second domain of the red sea turtle protease inhibitor. Clone p-Wp 52 has allowed the study of expression of whey phosphoprotein mRNA during functional differentiation of rat mammary gland and in mammary tumors. The whey phosphoprotein mRNA is detected during midpregnancy and lactation in the rat mammary gland but is barely detected in mammary tumors in which other milk protein mRNAs are expressed. The whey phosphoprotein gene in these tumors is hypermethylated, correlating with the reduced expression of this gene.

Amino Acid Sequence↗
PubMed1982

A 53-kilodalton protein common to chemically and virally transformed cells shows extensive sequence similarities between species.

A heat-stable DNA-binding protein with subunits of about 53 kilodaltons (kDal) was purified from two virally transformed human cell lines (Epstein-Barr virus-positive Raji and Namalwa) and two mouse tumor cell lines (methylcholanthrene-induced Meth A sarcoma and TA3 mammary carcinoma). All four 53kDal proteins showed closely related total amino acid compositions, similar peptide maps, and identical NH2-terminal amino acid sequences for 20 residues. These 53-kDal proteins are therefore evolutionarily highly conserved, independent of whether they originate from virally or chemically transformed cells. The NH2-terminal sequence and the protein chain as a whole are not hydrophobic; however, some unexpected residue distributions were observed. Comparisons with other proteins reveal no clear sequence similarity with known tumor antigen structures, homologous immunoglobulins, or some other proteins of known sequence. Epstein-Barr virus-determined nuclear antigen also appears to have a different NH2-terminal sequence. Thus, the results show that the 53-kDal proteins represent a unique protein type with little species variation; this finding suggests that these proteins must perform an important common function in different transformation systems.

Amino Acid Sequence↗
PubMed1982

Purification and biochemical properties of tumor-associated transplantation antigens from methylcholanthrene-induced murine sarcomas.

A tumor-associated transplantation antigen with an apparent molecular weight of 75,000 has been isolated from the cytosol of the BALB/c methylcholanthrene-induced sarcoma, Meth A. The antigen was purified either by preparative electrophoresis in the presence of NaDodSO4 or by immunoaffinity chromatography after hexylamine agarose chromatography, gel filtration, and hydroxylapatite chromatography. The 75-kilodalton (kDal) protein prepared by either of these methods effectively primed BALB/c mice to reject the Meth A tumor; such priming provided no protection against challenge by other independently derived sarcomas of BALB/c origin. A second protein, also 75 kDal, was isolated from the cytosol of the recently derived methylcholanthrene-induced sarcoma CI-4 by essentially the same chromatographic scheme. This protein also was immunogenic in the tumor rejection assay and provided protection only against CI-4 challenge. The antigens purified from the Meth A and CI-4 sarcomas appear to be closely related proteins. Both of them can be purified from the cytosol fraction and can be recognized by a rabbit antiserum prepared against the Meth A 75-kDal protein. The two proteins have approximately the same molecular weight, have similar but not identical amino acid compositions, and differ in their chromatographic behavior on hexylamine agarose and hydroxylapatite as well as in their isoelectric points. These results indicate that the individually specific transplantation antigens found in chemically induced sarcomas may be the products of a single multigene family or somatic derivatives of a single gene.

Amino Acids↗
PubMed1982

Assignment of the gene for beta 2-microglobulin (B2m) to mouse chromosome 2.

We have assigned the gene (B2m) coding for murine beta 2-microglobulin (B2M) to mouse chromosome 2 by using a novel panel of Chinese hamster-mouse somatic cell hybrid clones. Because of 35 independent primary hybrids used in this study were derived from two types of feral mice, each with a different combination of Robertsonian translocation chromosomes, as well as from mice with a normal complement of acrocentric chromosomes, analysis of 16 selected mouse enzyme markers provided data on the segregation of all 20 mouse chromosomes in these hybrids. Mouse B2M was identified in cell hybrids by immunoprecipitation with a species-specific anti-mouse B2M antiserum followed by two-dimensional polyacrylamide gel electrophoresis of the immunoprecipitated polypeptides. Enzyme analysis of the segregant clones excluded all chromosomes for B2m assignment except mouse chromosome 2, and karyotype analysis of nine informative hybrid clones confirmed the assignment of B2m to this chromosome. These results demonstrate that, in the mouse, as in man, B2m is not linked to the major histocompatibility or immunoglobulin loci.

Animals↗
PubMed1982

Multiple gamma-crystallins of the mouse lens: fractionation of mRNAs by cDNA cloning.

cDNAs made from polyadenylylated RNAs of the mouse lens were cloned by the G.C tailing procedure in the bacterial plasmid pBR322. Four recombinant DNAs containing gamma-crystallin sequences were identified by hybrid selection and translation. Sequence analysis of the in vivo-labeled gamma-crystallin polypeptides that cofocused isoelectrically with the hybrid-selected translation products established that the four cloned cDNAs were derived from mRNAs encoding gamma-crystallin polypeptides with similar NH2 termini. The cDNA clones had different restriction maps and could discriminate among the different gamma-crystallin mRNAs under stringent hybridization conditions. Under relaxed hybridization conditions, the cDNA clones cross-hybridized with all gamma-crystallin mRNAs, and even slightly with beta-crystallin mRNAs, as judged by in vitro translation. RNA blot hybridization showed that the mouse lens gamma-crystallin mRNAs are 840 +/- 100 nucleotides long. These data indicate that there are at least four similar gamma-crystallin mRNAs and suggest (but do not establish) the existence of a closely related family of gamma-crystallin genes.

Amino Acid Sequence↗
PubMed1981

Rapid removal of acetimidoyl groups from proteins and peptides. Applications to primary structure determination.

Methylamine buffers can be used for the rapid quantitative removal of acetimidoyl groups from proteins and peptides modified by treatment with ethyl or methyl acetimidate. The half-life for displacement of acetimidoyl groups from fully amidinated proteins incubated in 3.44 M-methylamine/HCl buffer at pH 11.5 and 25 degrees C was approx. 26 min; this half life is 29 times less than that observed in ammonia/HCl buffer under the same conditions of pH and amine concentration. Incubation of acetimidated proteins with methylamine for 4 h resulted in greater than 95% removal of acetimidoyl groups. No deleterious effects on primary structure were detected by amino acid analysis or by automated Edman degradation. Reversible amidination of lysine residues, in conjunction with tryptic digestion, has been successfully applied to the determination of the amino acid sequence of an acetimidated mouse immunoglobulin heavy chain peptide. The regeneration of amino groups in amidinated proteins and peptides by methylaminolysis makes amidination a valuable alternative to citraconoylation and maleoylation in structural studies.

Amino Acid Sequence↗
PubMed1981

The adenovirus hexon protein. The primary structure of the polypeptide and its correlation with the hexon gene.

The primary structure of the adenovirus hexon polypeptide has been determined by amino acid sequence studies of peptides from all regions of the molecule combined with sequence analysis of selected areas of its gene. The sequence presented contains 966 unique amino acid residues. Overlapping peptides recovered from CNBr cleavage and from digestions with proteolytic enzymes were analyzed, as well as DNA segments around sites for restriction endonucleases in the hexon gene. The primary structure is in good agreement with the total composition of the protein, with the compositions of individual CNBr fragments, and with known locations of restriction enzyme cleavage sites in the gene. Distinct regions of internal homology do not occur in the structure. The entire hexon polypeptide is encoded by a contiguous DNA sequence without intervening sequences.

Adenoviruses, Human↗
PubMed1981

Mouse beta 2-microglobulin cDNA clones: a screening procedure for cDNA clones corresponding to rare mRNAs.

We have isolated three cDNA clones for beta 2-microglobulin, the small subunit of the major histocompatibility antigens. beta 2-Microglobulin makes up less than 0.1% of mouse liver protein, and its mRNA is approximately 0.03% of liver poly(A)+ mRNA. The cDNA clones were identified by screening 1400 cDNA clones made from 9--10S mouse liver poly(A)+ mRNA. The procedure for screening the cDNA clones involved binding pooled plasmid DNA to nitrocellulose filters and testing the ability of each filter to select beta 2-microglobulin mRNA. The filter-selected mRNAs were assayed for their ability to direct the synthesis of beta 2-microglobulin in translation reactions in vitro. The isolated clones were shown by nucleotide sequence analysis to encode beta 2-microglobulin. The positive-selection--hybridization assay has been modified to facilitate the screening of large numbers of cDNA clones, and the modified assay should allow the isolation of cDNAs corresponding to any mRNA whose in vitro translation products can be immunoprecipitated. These modifications are of particular value in the isolation of cDNA clones corresponding to rare species of mRNA.

Amino Acid Sequence↗
PubMed1981

Control of expression of histocompatibility antigens (H-2) and beta 2-microglobulin in F9 teratocarcinoma stem cells.

Murine teratocarcinoma stem cells, unlike most other cell types, do not express major histocompatibility antigens. The steady-state levels of beta 2-microglobulin and H-2 mRNA from F9-derived teratocarcinoma stem and differentiated cells were examined by blot hybridization using cloned DNA probes specific for these mRNAs. No H-2- or beta 2-microglobulin-specific RNA was detected in F9 teratocarcinoma stem cells (clone 12-1); thus, F9 teratocarcinoma stem cells (clone 12-1) contain no more than 1/10 the H-2 and beta 2-microglobulin mRNAs of the differentiated daughter cells (clone 12-1a). We suggest that this regulation of major histocompatibility antigen expression is due to transcriptional control of the major histocompatibility antigen genes, H-2 and beta 2-microglobulin. The transcriptional regulation of these genes is accompanied by a change in their DNase I sensitivity. Normally, transcriptionally inactive genes are DNase I resistant, while active genes are DNase I sensitive. In contrast, the silent major histocompatibility antigen genes of teratocarcinoma stem cells are more DNase I sensitive than the active genes of the differentiated cells.

Beta-Globulins↗
PubMed1981

Parallel cross-reactivity patterns of 2 sets of antigenically distinct cytochrome c peptides: possible evidence for a presentational model of Ir gene function.

B10.A mice were immunized with either the carboxyl terminal peptide fragment 81-104 of pigeon cytochrome c or its acetimidyl derivative and an immune response was seen with strong preference for the immunogen. Strain distribution studies and blocking with an anti-Ia monoclonal antibody indicated that the same immune response (Ir) gene and restriction element were utilized in both responses. The specificity of the responses were evaluated by restimulating in vitro with a set of cytochrome c fragments from various species. Even though the derivatized and native fragments were poorly cross-reactive, the same phylogenetic pattern was seen when pigeon cytochrome c fragment 81-104 primed cells were tested with the set of underivatized fragments and when acetimidyl pigeon cytochrome c fragment 81-104 primed cells were tested with the same set of derivatized fragments. Primed cells from a 2nd major histocompatibility complex congenic strain of mice, B10.A(5R), displayed equivalent discrimination between derivatized and native forms but showed a markedly different phylogenetic pattern of cross-reactivity. These data indicate that the immune system recognizes 2 sites on the nominal antigen. One site, which accounts for the common hierarchy and is under Ir gene control, contains residues Gln-100, and possibly other carboxyl terminal residues. The 2nd site, which effects the distinction between native and derivatized fragments, contains at least 1 lysine other than at the carboxyl terminal. The implications of these data for theories of T cell recognition and Ir gene function are discussed.

Animals↗
PubMed1981

Lack of histocompatibility antigens on a murine ovarian teratocarcinoma.

We have attempted to generate in vitro lymphocytes cytotoxic to a widely studied model of ovarian cancer in C3HeB/FeJ mice. These attempts were unsuccessful with either syngeneic or allogeneic spleen cells. The following experimental results demonstrated that this murine ovarian tumor lacks histocompatibility antigens. (a) Tumor cells were not lysed by allogeneic lymphocytes presensitized to H-2k spleen cells. (b) Tumor cells did not specifically inhibit the cell-mediated lysis of H-2k spleen cells by presensitized allogeneic lymphocytes. (c) Histoincompatible (H-2b or H-2d) and syngeneic (H-2k) mice all died with identical tumor growth patterns within 25, 30, or 35 days following the i.p. inoculation of 10(6), 10(5), or 10(4) tumor cells, respectively. (d) Tumor cells were not lysed by an anti-H-2k antiserum and complement. (e) Absorption of the anti-H-2k antiserum with tumor cells did not decrease the cytotoxicity of the antiserum. (f) Competitive inhibition of a radioimmunoassay and polyacrylamide gel electrophoresis of immunoprecipitate of radiolabeled tumor extracts failed to demonstrate an H-2 heavy chain, although a normal amount of beta-microglobulin was present. This lack of histocompatibility antigens may explain the failure to generate lymphocytes cytotoxic to this tumor. Thus, this murine ovarian tumor, which has a serologically detectable tumor-associated antigen and can be cured by nonspecific immunotherapy, may provide an excellent model for the study of successful immunotherapy in the absence of histocompatibility antigens and associated cell-mediated reactions.

Animals↗
PubMed1980

Messenger RNA coding for the deleted heavy chain of mouse myeloma MOPC 47A immunoglobulin.

Mouse IgA 47A is an immunoglobulin variant whose heavy (alpha) chain has a molecular weight of 40,000 and lacks the entire CH3 domain. In order to understand better the mechanism by which the CH3 domain is lost, the 47A heavy chain mRNA has been partially purified. The mRNA migrated as a 17 S species on sucrose gradients and coded for the cell-free synthesis of only one serologically reactive protein with an apparent molecular weight of 42,000. Competitive inhibition of immunoprecipitation and tryptic peptide analysis of a cyanogen bromide fragment of the in vitro product revealed significant homology between the authentically secreted heavy chain and the respective cell-free synthesized, immunoprecipitated product. The heavy chain mRNA of 47A possessed a chain length of about 2200 nucleotides which is the same size as a normal alpha heavy chain mRNA, thus demonstrating that the protein deletion is not due to a large deletion at the mRNA level. The deletion in the 47A heavy chain may be due to a frameshift of a single nucleotide followed by premature chain termination.

Animals↗
PubMed1980

Complete amino acid sequence of a mouse immunoglobulin alpha chain (MOPC 511).

The complete amino acid sequence of the 432-residue heavy (alpha) chain of mouse myeloma MOPC 511 has been determined. The variable region of the alpha chain of IgA 511, a phosphocholine-binding protein, is highly homologous to that of the other phosphocholine-binding immunoglobulins. Comparison of the 511 alpha chain constant region with that of other mouse and human heavy chains shows that sequence divain. The CH3 domain disulfide bridge of the 511 alpha chain, for example, consists of only 28 amino acid residues compared to 60 residues for other chains and domains. Sequence divergences are alsos apparent at the CH2/CH3 domain boundary, an area where a number of frameshift mutations have occurred. One mutant, mouse IgA 47A, lacks the entire CH3 domain. Comparison of the 511 alppha chain with the 47A alpha chain reveals two noncconservative amino acid changes at the COOH terminus of the 47A chain, Ser-Gln for VAl-Thr in the 511 chain. These changes and the deletion of the CH3 domain can be explained by a single genetic event--namely, a frameshift mutation followed by premature chain termination. The remainder of the 47A constant region, including the hinge region, is identical to the 511 alpha chain, except for two conservative changes in the CH1 domain: serine-126 and theonine-197 in the 511 alpha chain are both replaced by alanine in the 47A chain.

Amino Acid Sequence↗