scholarly journals Isolation and oncogenic potential of a novel human src-like gene.

1986 ◽  
Vol 6 (12) ◽  
pp. 4195-4201 ◽  
Author(s):  
T Kawakami ◽  
C Y Pennington ◽  
K C Robbins
Keyword(s):  
Amino Acid ◽  
Protein Molecule ◽  
Nucleotide Sequence Analysis ◽  
Protein Kinase Activity ◽  
Amino Acid Residues ◽  
Amino Acid Homology ◽  
Oncogenic Potential ◽  
Coding Sequence ◽  
Amino Terminal ◽  
Sarcoma Virus

We have isolated cDNA molecules representing the complete coding sequence of a new human gene which is a member of the src family of oncogenes. Nucleotide sequence analysis revealed that this gene, termed slk, encoded a 537-residue protein which was 86% identical to the chicken proto-oncogene product, p60c-src, over a stretch of 191 amino acids at its carboxy terminus. In contrast, only 6% amino acid homology was observed within the amino-terminal 82 amino acid residues of these two proteins. It was possible to activate slk as a transforming gene by substituting approximately two-thirds of the slk coding sequence for an analogous region of the v-fgr onc gene present in Gardner-Rasheed feline sarcoma virus. The resulting hybrid protein molecule expressed in transformed cells demonstrated protein kinase activity with specificity for tyrosine residues.

Isolation and oncogenic potential of a novel human src-like gene

1986 ◽  
Vol 6 (12) ◽  
pp. 4195-4201
Author(s):  
T Kawakami ◽  
C Y Pennington ◽  
K C Robbins
Keyword(s):  
Amino Acid ◽  
Protein Molecule ◽  
Nucleotide Sequence Analysis ◽  
Protein Kinase Activity ◽  
Amino Acid Residues ◽  
Amino Acid Homology ◽  
Oncogenic Potential ◽  
Coding Sequence ◽  
Amino Terminal ◽  
Sarcoma Virus

We have isolated cDNA molecules representing the complete coding sequence of a new human gene which is a member of the src family of oncogenes. Nucleotide sequence analysis revealed that this gene, termed slk, encoded a 537-residue protein which was 86% identical to the chicken proto-oncogene product, p60c-src, over a stretch of 191 amino acids at its carboxy terminus. In contrast, only 6% amino acid homology was observed within the amino-terminal 82 amino acid residues of these two proteins. It was possible to activate slk as a transforming gene by substituting approximately two-thirds of the slk coding sequence for an analogous region of the v-fgr onc gene present in Gardner-Rasheed feline sarcoma virus. The resulting hybrid protein molecule expressed in transformed cells demonstrated protein kinase activity with specificity for tyrosine residues.

A Factor VIII Neutralizing Monoclonal Antibody and a Human Inhibitor Alloantibody Recognizing Epitopes in the C2 Domain Inhibit Factor VIII Binding to von Willebrand Factor and to Phosphatidylserine

1993 ◽  
Vol 69 (03) ◽  
pp. 240-246 ◽  
Author(s):  
Midori Shima ◽  
Dorothea Scandella ◽  
Akira Yoshioka ◽  
Hiroaki Nakai ◽  
Ichiro Tanaka ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Amino Acid ◽  
Factor Viii ◽  
Von Willebrand Factor ◽  
Light Chain ◽  
Amino Acid Residues ◽  
Amino Terminal ◽  
Neutralizing Monoclonal Antibody ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryA neutralizing monoclonal antibody, NMC-VIII/5, recognizing the 72 kDa thrombin-proteolytic fragment of factor VIII light chain was obtained. Binding of the antibody to immobilized factor VIII (FVIII) was completely blocked by a light chain-specific human alloantibody, TK, which inhibits FVIII activity. Immunoblotting analysis with a panel of recombinant protein fragments of the C2 domain deleted from the amino-terminal or the carboxy-terminal ends demonstrated binding of NMC-VIII/5 to an epitope located between amino acid residues 2170 and 2327. On the other hand, the epitope of the inhibitor alloantibody, TK, was localized to 64 amino acid residues from 2248 to 2312 using the same recombinant fragments. NMC-VIII/5 and TK inhibited FVIII binding to immobilized von Willebrand factor (vWF). The IC50 of NMC-VIII/5 for the inhibition of binding to vWF was 0.23 μg/ml for IgG and 0.2 μg/ml for F(ab)'2. This concentration was 100-fold lower than that of a monoclonal antibody NMC-VIII/10 which recognizes the amino acid residues 1675 to 1684 within the amino-terminal portion of the light chain. The IC50 of TK was 11 μg/ml by IgG and 6.3 μg/ml by F(ab)'2. Furthermore, NMC-VIII/5 and TK also inhibited FVIII binding to immobilized phosphatidylserine. The IC50 for inhibition of phospholipid binding of NMC-VIII/5 and TK (anti-FVIII inhibitor titer of 300 Bethesda units/mg of IgG) was 10 μg/ml.

Amino acid substitutions sufficient to convert the nontransforming p60c-src protein to a transforming protein

1986 ◽  
Vol 6 (12) ◽  
pp. 4155-4160
Author(s):  
J Y Kato ◽  
T Takeya ◽  
C Grandori ◽  
H Iba ◽  
J B Levy ◽  
...  
Keyword(s):  
Amino Acid ◽  
Biological Activities ◽  
Tumor Formation ◽  
Protein Kinase Activity ◽  
Focus Formation ◽  
Rous Sarcoma ◽  
Src Gene ◽  
Cellular Homolog ◽  
Sarcoma Virus ◽  
Hybrid Genes

We have previously shown that Rous sarcoma virus variants that carry the cellular homolog (c-src) of the viral src gene (v-src) do not transform chicken embryo fibroblasts. We also have shown that replacement of sequences upstream or downstream from the BglI site of the cellular src gene with the corresponding regions of v-src restored transforming activity to the hybrid genes. Since there are only six amino acid changes between p60c-src and p60v-src within the sequences upstream from BglI, we constructed chimeric molecules involving v-src and c-src to determine the effect of each amino acid substitution on the biological activities of the gene product. We found that the change from Thr to Ile at position 338 or the replacement of a fragment of c-src containing Gly-63, Arg-95, and Thr-96 with a corresponding fragment of v-src containing Asp-63, Trp-95, and Ile-96 converted p60c-src into a transforming protein by the criteria of focus formation, anchorage-independent growth, and tumor formation in newborn chickens. These mutations also resulted in elevation of the protein kinase activity of p60c-src.

Isolation and DNA sequence of ADH3, a nuclear gene encoding the mitochondrial isozyme of alcohol dehydrogenase in Saccharomyces cerevisiae

1985 ◽  
Vol 5 (11) ◽  
pp. 3024-3034
Author(s):  
E T Young ◽  
D Pilgrim
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Nucleotide Sequence ◽  
Alcohol Dehydrogenase ◽  
Nuclear Gene ◽  
Amino Acid Identity ◽  
Leader Peptide ◽  
Nucleotide Sequence Analysis ◽  
Amino Terminal ◽  
Cloned Gene

The Saccharomyces cerevisiae nuclear gene, ADH3, that encodes the mitochondrial alcohol dehydrogenase isozyme ADH III was cloned by virtue of its nucleotide homology to ADH1 and ADH2. Both chromosomal and plasmid-encoded ADH III isozymes were repressed by glucose and migrated heterogeneously on nondenaturing gels. Nucleotide sequence analysis indicated 73 and 74% identity for ADH3 with ADH1 and ADH2, respectively. The amino acid identity between the predicted ADH III polypeptide and ADH I and ADH II was 79 and 80%, respectively. The open reading frame encoding ADH III has a highly basic 27-amino-acid amino-terminal extension relative to ADH I and ADH II. The nucleotide sequence of the presumed leader peptide has a high degree of identity with the untranslated leader regions of ADH1 and ADH2 mRNAs. A strain containing a null allele of ADH3 did not have a detectably altered phenotype. The cloned gene integrated at the ADH3 locus, indicating that this is the structural gene for ADH III.

scholarly journals Identification of Epitope and Surface-Exposed Domains of Shigella flexneri Invasion Plasmid Antigen D (IpaD)

1998 ◽  
Vol 66 (5) ◽  
pp. 1999-2006 ◽  
Author(s):  
K. Ross Turbyfill ◽  
Jennifer A. Mertz ◽  
Corey P. Mallett ◽  
Edwin V. Oaks
Keyword(s):  
Amino Acid ◽  
Shigella Flexneri ◽  
Serum Antibody ◽  
Terminal Region ◽  
Virulence Plasmid ◽  
Amino Acid Residues ◽  
Unique Region ◽  
Amino Terminal ◽  
Convalescent Phase ◽  
Carboxyl Terminal

ABSTRACT Transport and surface expression of the invasion plasmid antigens (Ipa proteins) is an essential trait in the pathogenicity ofShigella spp. In addition to the type III protein secretion system encoded by the mxi/spa loci on the large virulence plasmid, transport of IpaB and IpaC into the surrounding medium is modulated by IpaD. To characterize the structural topography of IpaD, the Geysen epitope-mapping system was used to identify epitopes recognized by surface-reactive monoclonal and polyclonal antibodies produced against purified recombinant IpaD or synthetic IpaD peptides. Surface-exposed epitopes of IpaD were confined to the first 180 amino acid residues, whereas epitopes in the carboxyl-terminal half were not exposed on the Shigella surface. By using convalescent-phase sera from 10 Shigella flexneri-infected monkeys, numerous epitopes were mapped within a surface-exposed region of IpaD between amino acid residues 14 and 77. Epitopes were also identified in the carboxyl-terminal half of IpaD with a few convalescent-phase sera. Comparison of IpaD epitope sequences withSalmonella SipD sequences indicated that very similar epitopes may exist in the carboxyl-terminal region of each protein whereas the IpaD epitopes in the surface-exposed amino-terminal region were unique for the Shigella protein. Although the IpaD and SipD homologs may play similar roles in transport, the dominant serum antibody response to IpaD is against the unique region of this protein exposed on the surface of the pathogen.

Characterization of the sulfated glycopeptide of chicken pepsinogen

1982 ◽  
Vol 47 (2) ◽  
pp. 709-718 ◽  
Author(s):  
Miroslav Baudyš ◽  
Vladimír Kostka ◽  
Karel Grüner ◽  
Jan Pohl
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Protein Molecule ◽  
Amino Acid Residues ◽  
Terminal Sequence ◽  
Sugar Moiety ◽  
Pepsinogen A ◽  
Identical Amino Acid Sequence ◽  
High Voltage Electrophoresis

S-sulfonated chicken pepsinogen was digested with TPCK-trypsin; large tryptic peptides, separated on Sephadex G-25 fine, were subjected to additional cleavage with α-chymotrypsin. The hold-up fraction of the chymotryptic digest from the Sephadex G-25 column, was resolved by high voltage electrophoresis. The three most acidic zones contained glycopeptides of identical amino acid sequence Val-Ser-Thr-Asn-Glu-Thr-Val-Tyr, yet differed in the composition of the sugar moiety. These glycopeptides, moreover, bear different numbers of sulfate groups which enabled the resolution of the peptides. The most acidic glycopeptide contains 7 glucosamine residues, 3 mannose residues and 5 sulfate groups, the second one 6 glucosamine residues, 3 mannose residues and 4 sulfate groups and the slowest, minority glycopeptide, 5 glucosamine residues, 2 mannose residues and 2 sulfate groups. The entire sugar moiety is attached to one of the chain viaasparagine. In other experiments the glycopeptides were also isolated from the thermolytic digest of chicken pepsin; their C-terminal sequence was shorter by two amino acid residues. The tentative assignment of the glycopeptides to the amino acid sequence of pepsinogen resulted from the analysis of the limited tryptic digest of the whole protein molecule. Chicken pepsinogen is glycosylated at the site of the chain occupied by a phosphoserine residue in hog pepsinogen A.

Purification, properties, and partial amino acid sequence of chitinase from a marine Alteromonas sp. strain O-7

1992 ◽  
Vol 38 (9) ◽  
pp. 891-897 ◽  
Author(s):  
Hiroshi Tsujibo ◽  
Yukio Yoshida ◽  
Katsushiro Miyamoto ◽  
Chiaki Imada ◽  
Yoshiro Okami ◽  
...  
Keyword(s):  
Amino Acid ◽  
Marine Bacterium ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Molecular Size ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Amino Acid Residues ◽  
Amino Terminal

Chitinase (EC 3.2.1.14) was isolated from the culture supernatant of a marine bacterium, Alteromonas sp. strain O-7. The enzyme (Chi-A) was purified by anion-exchange chromatography (DEAE-Toyopearl 650 M) and gel filtration (Sephadex G-100). The purified enzyme showed a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The molecular size and pI of Chi-A were 70 kDa and 3.9, respectively. The optimum pH and temperature of Chi-A were 8.0 and 50 °C, respectively. Chi-A was stable in the range of pH 5–10 up to 40 °C. Among the main cations, such as Na+, K+, Mg2+, and Ca2+, contained in seawater, Mg2+ stimulated Chi-A activity. N-Bromosuccinimide and 2-hydroxy-5-nitrobenzyl bromide inhibited Chi-A activity. The amino-terminal 27 amino acid residues of Chi-A were sequenced. This enzyme showed sequence homology with chitinases from terrestrial bacteria such as Serratia marcescens QMB1466 and Bacillus circulons WL-12. Key words: marine bacterium, Alteromonas sp., chitinase.

scholarly journals Cloning and structural characterization of the genes coding for adenosylcobalamin-dependent methylmalonyl-CoA mutase from Propionibacterium shermanii

1989 ◽  
Vol 260 (2) ◽  
pp. 345-352 ◽  
Author(s):  
E N Marsh ◽  
N McKie ◽  
N K Davis ◽  
P F Leadlay
Keyword(s):  
Amino Acid ◽  
Alpha Subunit ◽  
Nucleotide Sequence Analysis ◽  
Homologous Region ◽  
Amino Acid Residues ◽  
Upstream Gene ◽  
Ancestral Gene ◽  
Propionibacterium Shermanii ◽  
Hybridization Probes

The structural genes coding for both subunits of adenosylcobalamin-dependent methylmalonyl-CoA mutase from the Gram-positive bacterium Propionibacterium shermanii have been cloned, with the use of synthetic oligonucleotides as primary hybridization probes. The genes are closely linked and are transcribed in the same direction. Nucleotide sequence analysis of 4.5 kb of DNA encompassing both genes allowed us to infer the complete amino acid sequence of the two subunits: the beta-subunit is the product of the upstream gene, and consists of 638 amino acid residues (Mr 69465) and the alpha-subunit consists of 728 amino acid residues (Mr 80,147). There is a very close structural homology between the two subunits, reflecting the probable duplication of a common ancestral gene. A sequence present only in the alpha-subunit is significantly homologous to a portion of the sequence of the methylmalonyl-CoA-binding subunit of transcarboxylase from P. shermanii [Samols, Thornton, Murtif, Kumar, Haase & Wood (1988) J. Biol. Chem. 263, 6461-6464], and this homologous region may form part of the CoA ester-binding site in both enzymes.

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