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Papers by Charles Hasemann
Journal of Immunology, Nov 1, 1991
The elucidation of the structural basis for expression of the cross-reactive Id of the A strain m... more The elucidation of the structural basis for expression of the cross-reactive Id of the A strain mouse (CRIA) in response to the hapten p-azophenylarsonate has been the object of considerable research effort. Most conclusions regarding the amino acids involved in Id expression have been inferential, based on comparisons of amino acid sequences, chain recombination experiments, or chemical modification of particular amino acids. To more rigorously designate the amino acids critical to the phenotype of the CRIA, a system for the expression and directed mutation of antibody molecules was developed. Based on the baculovirus expression of foreign proteins in cultured insect cells, functional antibodies can be produced at very high levels in vitro. By the process of oligonucleotide-directed mutagenesis, a series of specific amino acid changes were introduced into both the H and L chains of a prototype CRIA expressing antibody molecule. Analysis of the gain or loss of polyclonal Id and each of several monoclonal idiotopes has allowed us to identify more precisely the amino acids responsible for expression of the CRIA. Thus we have shown that expression of the CRIA is equally dependent on amino acids in the second and third CDR of the H chain. Furthermore, the idiotopes studied surround the Ag-binding site and clearly involve multiple interactions in several of the L and H chain CDR.
with specic help available everywhere you see the i O symbol. The following versions of software ... more with specic help available everywhere you see the i O symbol. The following versions of software and data (see references i O) were used in the production of this report:
Journal of Bacteriology, Jul 15, 1999
Proceedings of the National Academy of Sciences of the United States of America, Jun 15, 1992
Crystallization and preliminary x-ray diffraction analysis of P450terp and the hemoprotein domain... more Crystallization and preliminary x-ray diffraction analysis of P450terp and the hemoprotein domain of P450BM-3, enzymes belonging to two distinct classes of the cytochrome P450 superfamily
Humana Press eBooks, 1987
This paper will attempt to provide an overview as to how recombinant DNA and protein chemical tec... more This paper will attempt to provide an overview as to how recombinant DNA and protein chemical techniques work hand-in-glove in order to relatively easily determine complete structural information concerning a series of molecules. Emphasis will be placed on immunoglobulin sequencing utilizing picomole amounts of murine and human hybridomas to determine the N-terminal one third of both heavy and light polypeptide chains and the use of oligonucleotide primers for mRNA sequencing of the same molecules. The overlaps in the center of the variable regions provide assurance that the two techniques allow the complete primary structure of immunoglobulin variable regions to be deduced rather rapidly. Techniques for fragmentation are unnecessary (which would be required if only protein chemistry techniques were used) and second and third “primings” are not required (as would be necessitated by relying solely on mRNA sequencing techniques). These studies have resulted in the complete structural analysis of a series of human and murine hybridomas from extremely small amounts of ascites fluid, culture fluid and cell pellets.
Journal of Bacteriology, Jul 1, 1999
The Journal of Immunology
The elucidation of the structural basis for expression of the cross-reactive Id of the A strain m... more The elucidation of the structural basis for expression of the cross-reactive Id of the A strain mouse (CRIA) in response to the hapten p-azophenylarsonate has been the object of considerable research effort. Most conclusions regarding the amino acids involved in Id expression have been inferential, based on comparisons of amino acid sequences, chain recombination experiments, or chemical modification of particular amino acids. To more rigorously designate the amino acids critical to the phenotype of the CRIA, a system for the expression and directed mutation of antibody molecules was developed. Based on the baculovirus expression of foreign proteins in cultured insect cells, functional antibodies can be produced at very high levels in vitro. By the process of oligonucleotide-directed mutagenesis, a series of specific amino acid changes were introduced into both the H and L chains of a prototype CRIA expressing antibody molecule. Analysis of the gain or loss of polyclonal Id and each...
This article cites 29 articles, 14 of which can be accessed free
Methods in Protein Sequence Analysis · 1986, 1987
This paper will attempt to provide an overview as to how recombinant DNA and protein chemical tec... more This paper will attempt to provide an overview as to how recombinant DNA and protein chemical techniques work hand-in-glove in order to relatively easily determine complete structural information concerning a series of molecules. Emphasis will be placed on immunoglobulin sequencing utilizing picomole amounts of murine and human hybridomas to determine the N-terminal one third of both heavy and light polypeptide chains and the use of oligonucleotide primers for mRNA sequencing of the same molecules. The overlaps in the center of the variable regions provide assurance that the two techniques allow the complete primary structure of immunoglobulin variable regions to be deduced rather rapidly. Techniques for fragmentation are unnecessary (which would be required if only protein chemistry techniques were used) and second and third “primings” are not required (as would be necessitated by relying solely on mRNA sequencing techniques). These studies have resulted in the complete structural analysis of a series of human and murine hybridomas from extremely small amounts of ascites fluid, culture fluid and cell pellets.
This article cites 21 articles, 8 of which can be accessed free at:
Infection and Immunity, 1989
Sequencing techniques for single- and double-stranded DNA were used to determine the nucleotide s... more Sequencing techniques for single- and double-stranded DNA were used to determine the nucleotide sequence of the gene encoding P2, the major outer membrane (porin) protein of Haemophilus influenzae type b (Hib). The open reading frame encoding the P2 protein comprised 361 amino acid codons. Comparison of the inferred amino acid sequence with data obtained by amino acid sequencing of the N terminus of the mature or fully processed P2 protein revealed that this protein has a signal peptide composed of 20 amino acids. N-terminal amino acid sequencing of tryptic peptides derived from purified P2 allowed direct identification of 158 of the 341 amino acids in the fully processed P2 protein; there was 100% correlation between these amino acid sequences and that inferred from the nucleotide sequence. The amino acid sequence of Hib P2 protein had 23 to 25% homology with the sequence of the OmpF porin of Escherichia coli and with that of the Neisseria gonorrhoeae porin P.IA. Codon usage in the...
This is a Full wwPDB X-ray Structure Validation Report for a publicly released PDB entry.
Protein Science
Diffraction-quality crystals of the bifunctional enzyme fructose 6-phosphate, 2-kinase:fructose 2... more Diffraction-quality crystals of the bifunctional enzyme fructose 6-phosphate, 2-kinase:fructose 2,6-bisphosphatase from rat testis have been obtained. The crystals were grown in the presence of ATP gamma S, fructose 6-phosphate, the detergent n-octylglucoside, and the precipitant polyethylene glycol 4000. The crystals have the symmetry of the trigonal space group P31/221 with a = b = 83.0 A and c = 130.6 A. Flash-frozen crystals diffract to beyond 2.2 A, and native data have been collected.
Nature Structural & Molecular Biology, 2004
A typographical error was inadvertently introduced in Figure 1a. The corrected chemical structure... more A typographical error was inadvertently introduced in Figure 1a. The corrected chemical structure is shown below. The authors apologize for any inconvenience this may have caused.
European Journal of Immunology, 1989
Molecular characterization of the VH region of murine autoantibodies from neonatal and adult BALB... more Molecular characterization of the VH region of murine autoantibodies from neonatal and adult BALBk mice* We report on the molecular characterization of the heavy (H) chain variable (V) region of two murine autoantibodies reacting with a conventional self antigen, thyroglobulin (Tg), originated from unimmunized/unstimulated neonatal mice (clone B10H2) and from an adult hyperimmunized mouse (clone 62), respectively. Serologically, both hybridoma antibodies express the same idiotype (Id). By cloning and sequencing we demonstrated that their VH regions are encoded by identical VDJ gene elements including the VD and DJ joints. This VH belongs to the VH 7183 gene family, the most 3'-end proximal family in the murine genome. The D segment is unique and only 50% similar to any murine D segment. The J segment utilized is germ-line J d. The cloned DNA rearrangement was transfected into the J558L myeloma cells and the secreted antibody was found to express the reference Id on the H chain, hence proving that the productive VDJ rearrangement had been identified. These results show that a spontaneously arising and an antigen-induced autoantibody use an identical VDJ gene rearrangement and that after hyperimmunization somatic mutation did not occur. The significance of this finding with respect to ontogeny of the B cell repertoire is discussed.
Journal of Immunology, Nov 1, 1991
The elucidation of the structural basis for expression of the cross-reactive Id of the A strain m... more The elucidation of the structural basis for expression of the cross-reactive Id of the A strain mouse (CRIA) in response to the hapten p-azophenylarsonate has been the object of considerable research effort. Most conclusions regarding the amino acids involved in Id expression have been inferential, based on comparisons of amino acid sequences, chain recombination experiments, or chemical modification of particular amino acids. To more rigorously designate the amino acids critical to the phenotype of the CRIA, a system for the expression and directed mutation of antibody molecules was developed. Based on the baculovirus expression of foreign proteins in cultured insect cells, functional antibodies can be produced at very high levels in vitro. By the process of oligonucleotide-directed mutagenesis, a series of specific amino acid changes were introduced into both the H and L chains of a prototype CRIA expressing antibody molecule. Analysis of the gain or loss of polyclonal Id and each of several monoclonal idiotopes has allowed us to identify more precisely the amino acids responsible for expression of the CRIA. Thus we have shown that expression of the CRIA is equally dependent on amino acids in the second and third CDR of the H chain. Furthermore, the idiotopes studied surround the Ag-binding site and clearly involve multiple interactions in several of the L and H chain CDR.
with specic help available everywhere you see the i O symbol. The following versions of software ... more with specic help available everywhere you see the i O symbol. The following versions of software and data (see references i O) were used in the production of this report:
Journal of Bacteriology, Jul 15, 1999
Proceedings of the National Academy of Sciences of the United States of America, Jun 15, 1992
Crystallization and preliminary x-ray diffraction analysis of P450terp and the hemoprotein domain... more Crystallization and preliminary x-ray diffraction analysis of P450terp and the hemoprotein domain of P450BM-3, enzymes belonging to two distinct classes of the cytochrome P450 superfamily
Humana Press eBooks, 1987
This paper will attempt to provide an overview as to how recombinant DNA and protein chemical tec... more This paper will attempt to provide an overview as to how recombinant DNA and protein chemical techniques work hand-in-glove in order to relatively easily determine complete structural information concerning a series of molecules. Emphasis will be placed on immunoglobulin sequencing utilizing picomole amounts of murine and human hybridomas to determine the N-terminal one third of both heavy and light polypeptide chains and the use of oligonucleotide primers for mRNA sequencing of the same molecules. The overlaps in the center of the variable regions provide assurance that the two techniques allow the complete primary structure of immunoglobulin variable regions to be deduced rather rapidly. Techniques for fragmentation are unnecessary (which would be required if only protein chemistry techniques were used) and second and third “primings” are not required (as would be necessitated by relying solely on mRNA sequencing techniques). These studies have resulted in the complete structural analysis of a series of human and murine hybridomas from extremely small amounts of ascites fluid, culture fluid and cell pellets.
Journal of Bacteriology, Jul 1, 1999
The Journal of Immunology
The elucidation of the structural basis for expression of the cross-reactive Id of the A strain m... more The elucidation of the structural basis for expression of the cross-reactive Id of the A strain mouse (CRIA) in response to the hapten p-azophenylarsonate has been the object of considerable research effort. Most conclusions regarding the amino acids involved in Id expression have been inferential, based on comparisons of amino acid sequences, chain recombination experiments, or chemical modification of particular amino acids. To more rigorously designate the amino acids critical to the phenotype of the CRIA, a system for the expression and directed mutation of antibody molecules was developed. Based on the baculovirus expression of foreign proteins in cultured insect cells, functional antibodies can be produced at very high levels in vitro. By the process of oligonucleotide-directed mutagenesis, a series of specific amino acid changes were introduced into both the H and L chains of a prototype CRIA expressing antibody molecule. Analysis of the gain or loss of polyclonal Id and each...
This article cites 29 articles, 14 of which can be accessed free
Methods in Protein Sequence Analysis · 1986, 1987
This paper will attempt to provide an overview as to how recombinant DNA and protein chemical tec... more This paper will attempt to provide an overview as to how recombinant DNA and protein chemical techniques work hand-in-glove in order to relatively easily determine complete structural information concerning a series of molecules. Emphasis will be placed on immunoglobulin sequencing utilizing picomole amounts of murine and human hybridomas to determine the N-terminal one third of both heavy and light polypeptide chains and the use of oligonucleotide primers for mRNA sequencing of the same molecules. The overlaps in the center of the variable regions provide assurance that the two techniques allow the complete primary structure of immunoglobulin variable regions to be deduced rather rapidly. Techniques for fragmentation are unnecessary (which would be required if only protein chemistry techniques were used) and second and third “primings” are not required (as would be necessitated by relying solely on mRNA sequencing techniques). These studies have resulted in the complete structural analysis of a series of human and murine hybridomas from extremely small amounts of ascites fluid, culture fluid and cell pellets.
This article cites 21 articles, 8 of which can be accessed free at:
Infection and Immunity, 1989
Sequencing techniques for single- and double-stranded DNA were used to determine the nucleotide s... more Sequencing techniques for single- and double-stranded DNA were used to determine the nucleotide sequence of the gene encoding P2, the major outer membrane (porin) protein of Haemophilus influenzae type b (Hib). The open reading frame encoding the P2 protein comprised 361 amino acid codons. Comparison of the inferred amino acid sequence with data obtained by amino acid sequencing of the N terminus of the mature or fully processed P2 protein revealed that this protein has a signal peptide composed of 20 amino acids. N-terminal amino acid sequencing of tryptic peptides derived from purified P2 allowed direct identification of 158 of the 341 amino acids in the fully processed P2 protein; there was 100% correlation between these amino acid sequences and that inferred from the nucleotide sequence. The amino acid sequence of Hib P2 protein had 23 to 25% homology with the sequence of the OmpF porin of Escherichia coli and with that of the Neisseria gonorrhoeae porin P.IA. Codon usage in the...
This is a Full wwPDB X-ray Structure Validation Report for a publicly released PDB entry.
Protein Science
Diffraction-quality crystals of the bifunctional enzyme fructose 6-phosphate, 2-kinase:fructose 2... more Diffraction-quality crystals of the bifunctional enzyme fructose 6-phosphate, 2-kinase:fructose 2,6-bisphosphatase from rat testis have been obtained. The crystals were grown in the presence of ATP gamma S, fructose 6-phosphate, the detergent n-octylglucoside, and the precipitant polyethylene glycol 4000. The crystals have the symmetry of the trigonal space group P31/221 with a = b = 83.0 A and c = 130.6 A. Flash-frozen crystals diffract to beyond 2.2 A, and native data have been collected.
Nature Structural & Molecular Biology, 2004
A typographical error was inadvertently introduced in Figure 1a. The corrected chemical structure... more A typographical error was inadvertently introduced in Figure 1a. The corrected chemical structure is shown below. The authors apologize for any inconvenience this may have caused.
European Journal of Immunology, 1989
Molecular characterization of the VH region of murine autoantibodies from neonatal and adult BALB... more Molecular characterization of the VH region of murine autoantibodies from neonatal and adult BALBk mice* We report on the molecular characterization of the heavy (H) chain variable (V) region of two murine autoantibodies reacting with a conventional self antigen, thyroglobulin (Tg), originated from unimmunized/unstimulated neonatal mice (clone B10H2) and from an adult hyperimmunized mouse (clone 62), respectively. Serologically, both hybridoma antibodies express the same idiotype (Id). By cloning and sequencing we demonstrated that their VH regions are encoded by identical VDJ gene elements including the VD and DJ joints. This VH belongs to the VH 7183 gene family, the most 3'-end proximal family in the murine genome. The D segment is unique and only 50% similar to any murine D segment. The J segment utilized is germ-line J d. The cloned DNA rearrangement was transfected into the J558L myeloma cells and the secreted antibody was found to express the reference Id on the H chain, hence proving that the productive VDJ rearrangement had been identified. These results show that a spontaneously arising and an antigen-induced autoantibody use an identical VDJ gene rearrangement and that after hyperimmunization somatic mutation did not occur. The significance of this finding with respect to ontogeny of the B cell repertoire is discussed.