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K L Knight

Publications and source records attributed to K L Knight.

At least 109 records · Page 6Linked to original sources
PubMed1989

Evolutionary conservation of splice sites in sterile C mu transcripts and of immunoglobulin heavy chain (IgH) enhancer region sequences.

The immunoglobulin JHC mu intron was cloned from genomic DNA of a VHa3 rabbit and a 1257 bp sequence which contains conserved enhancer and splice sites was determined. From positions 315 to 1257, there is approximately 72 and 67% similarity to available sequences of man and mouse, respectively (counting gaps as single changes at single positions). In earlier studies of rabbit cDNAs encoding immunoglobulin heavy chains, we found a C mu-encoding cDNA clone (pB3) derived from splenic mRNA of a Trypanosome-hyperimmunized rabbit (VHa1) which lacked VH, DH or JH sequences and had an unknown sequence 5' of that encoding C mu. Comparison of this cDNA sequence with the present cloned genomic DNA sequence has now revealed that the start of cDNA pB3 corresponds to a position 80 base pairs 3' of the conserved octamer motif of the rabbit heavy chain enhancer. This mRNA was spliced to the acceptor site of C mu using a donor site which was 635 bp 3' of the enhancer octanucleotide. Our sequence of pB3 indicates that in rabbit as in mouse, a "nontron" (33 stop codons in three reading frames) can be formed utilizing a conserved splice site to produce a spliced transcript. The presence of evolutionarily conserved splice donor sites in the intron sequences of rabbit, mouse and man suggests a functional role during B cell ontogeny.

Animals↗
PubMed1989

Expression of 12 rabbit IgA C alpha genes as chimeric rabbit-mouse IgA antibodies.

Serologic analysis of rabbit secretory IgA initially identified two subclasses of IgA, IgA-f and IgA-g. Recent molecular genetic studies have resulted in the identification and cloning of 13 genes encoding the constant region (C) of rabbit IgA heavy chains. Each of these 13 C alpha genes, C alpha 1-C alpha 13, was subcloned into an expression vector containing the VDJ (V, variable; D, diversity; J, joining) gene of a dansyl (DNS)-binding hybridoma antibody. The alpha heavy-chain constructs were transfected into SP2/0 cells producing murine light chains with specificity for DNS. Of the 13 resulting transfectomas, 12 were shown by ELISA to secrete DNS-binding chimeric rabbit-mouse IgA molecules. By immunoblot analysis, the 12 IgA-producing transfectomas were shown to secrete alpha chains ranging in size from 60 to 72 kDa. These data suggest that rabbit IgA may be composed of as many as 12 IgA isotypes. This is in marked contrast to mouse and human, in which only 1 and 2 IgA isotypes, respectively, are found. Serologic analyses, using anti-IgA-f and anti-IgA-g alloantisera, revealed that 11 of the 12 transfectoma IgAs reacted with anti-IgA-f and not with anti-IgA-g antibodies and that one reacted with anti-IgA-g and not with anti-IgA-f antibodies. Each of the IgA-producing transfectomas was cocultured with a Madin-Darby canine kidney cell line expressing the rabbit polymeric immunoglobulin receptor, and the transcytosed IgA antibodies were analyzed by immunoblots to determine whether they associated with secretory component (SC) through covalent or noncovalent interactions. Each of the 11 IgA-f isotypes was shown to bind SC by a disulfide linkage, whereas the single IgA-g isotype appeared to bind SC through noncovalent interactions only.

Animals↗
PubMed1989

Rabbit major histocompatibility complex. III. Multiple class II DR beta genes and restriction fragment length polymorphism of the class II alpha and beta genes.

Several class II alpha and beta chain genes of the rabbit MHC have been cloned and classified into three distinct subregions, R-DP, R-DQ and R-DR, based on their homology to the corresponding HLA-DP, -DQ and -DR genes. The organization of the rabbit MHC class II genes has now been studied in greater detail by analysing genomic DNA of an inbred III/J strain and several other RLA-D homozygous rabbits, with DNA probes derived from cloned R-DR beta genes. Eight previously cloned R-DR beta genes were shown to be allelic forms of five R-DR beta loci. Genomic blot analyses of DNA from seven rabbits homozygous for different RLA haplotypes revealed that the germline contains a total of approximately seven class II beta genes, one DQ beta, one DP beta and five DR beta. Extensive allelic polymorphism was identified by RFLP analysis using DQ and DR probes; limited RFLP was observed with DP probes. RFLP analyses allowed us to distinguish two haplotypes which had not been previously distinguished by MLR. Such RFLP analyses will be useful for identifying MHC 'compatible' rabbits for various immunobiological studies, including transplantation.

Animals↗
PubMed1988

Genetic engineering of bovine Ig. Construction and characterization of hapten-binding bovine/murine chimeric IgE, IgA, IgG1, IgG2, and IgG3 molecules.

A bovine recombinant phage library was constructed and screened with rabbit S mu and human C gamma probes. IgM, IgA, and IgE H chain constant region (CH) genes were isolated with the rabbit S mu probe and three IgG CH genes were isolated with the C gamma probe. The CH genes were individually cloned into an expression vector which contained a murine VDJ gene cloned from a hybridoma producing anti-dansyl hapten antibody. The resulting constructs were transfected into murine hybridoma cells producing L chain of the anti-dansyl antibody and stable transfectomas secreting chimeric bovine-murine IgA, IgE, or IgG subclass anti-dansyl antibodies were obtained. The chimeric antibodies, immunoprecipitated with Ag or with anti-bovine H chain antibodies, were analyzed by SDS-PAGE and were shown to contain H and L chains of expected size. Of the three chimeric antibodies derived from the C gamma genes, one reacted with anti-IgG1 antibody, another reacted with anti-IgG2 antibody and the third did not react with either anti-IgG1 or anti-IgG2. This third IgG appears to represent a "new" subclass of bovine IgG, IgG3. Southern blot analysis indicated that the bovine genome contains a fourth C gamma gene. These experiments demonstrate the usefulness of molecular genetic techniques for the isolation and characterization of Ig which are not readily purified from biologic fluids. These techniques will be useful for isolation and characterization of Ig genes from other outbred mammals.

Animals↗
PubMed1988

Rabbit MHC. II. Sequence analysis of the R-DP alpha- and beta-genes.

Molecular genetics studies have recently revealed complexity in the MHC class II region that has not been detected by previous serologic and genetic studies. In humans, three subregions, DP, DQ, and DR, of the class II genes as well as the DZ alpha and DO beta genes, have been extensively characterized. Although homologs of these human genes were identified in many species, their expressibility has not been well defined in species other than the mouse. We have previously cloned the rabbit homologs of the HLA-DP alpha and beta genes whose protein products had never been detected. The sequences of rabbit DP alpha 1 and DP beta genes are reported herein and they indicate that the rabbit DP genes encode functional alpha- and beta-chains. Unfavorable nucleotides surrounding the first AUG codon may, however, reduce the translational efficiency of the R-DP beta mRNA and explain the difficulty in generating serologic reagents specific for rabbit DP molecules. A complex mutation in the beta 1 domain of the R-DP beta gene was similar to the one found in the H-2A beta 1 gene of five strains of mice. The origin of this mutation is discussed.

Amino Acid Sequence↗
PubMed1988

The Mnt repressor of bacteriophage P22: role of C-terminal residues in operator binding and tetramer formation.

A set of C-terminal deletion mutants of the Mnt repressor of bacteriophage P22 has been constructed, and the corresponding truncated proteins have been purified. A truncated protein lacking the three C-terminal residues, Lys80-Thr81-Thr82, binds operator DNA with an affinity near wild type and has a normal tetrameric structure. Loss of the next residue, Lys79, causes a 600-fold decrease in operator affinity, but the truncated protein is still tetrameric. Further sequential deletions of Tyr78 and Leu77 cause modest decreases in operator affinity, but the truncated proteins are now dimeric. These results indicate that Lys79 is an important determinant of the high affinity of Mnt repressor for operator DNA and that Tyr78 is an important determinant of tetramer formation by Mnt repressor.

Amino Acid Sequence↗
PubMed1988

Partial molecular genetic map of the rabbit VH chromosomal region.

Thirty VH-containing cosmid clones, isolated from rabbit germ-line DNA libraries, were restriction mapped and shown to contain approximately 100 VH genes in 765-kb of DNA. Twenty-two of the cosmid clones were grouped into seven distinct clusters. The VH genes were separated by an average of 8 kb, although some were separated by less than 3 kb. Comparison of the nucleotide sequences of two of these VH genes with the sequences of another 11 VH genes showed that they were all generally more than 80% homologous suggesting that rabbit VH genes are members of one highly homologous gene family. Most rabbit Ig molecules have the VH allotypic specificities a1, a2, or a3 and are designated VHa-positive. A small number (less than 30%) of Ig molecules lack these VHa allotypic specificities and are designated VHa-negative. The VH containing cosmid clones were hybridized with synthetic oligomer probes designed to be specific for genes encoding VHa-positive or VHa-negative molecules. At least 50% of the germ-line VH genes hybridized with the VHa-negative oligomer and thus presumably encode VHa-negative molecules; as few as 15% of the genes could be identified as encoding VHa-positive molecules based on hybridization with the VHa-positive oligomer. Approximately 35% of the VH genes did not hybridize with either oligomer and could not be classified as VHa-negative or VHa-positive. We propose that the predominance of serum VHa-positive molecules, in contrast to the predominance of VHa-negative encoding germ-line genes, may reflect preferential usage of a few germline VH genes. The implications of this idea toward explaining the allelic inheritance of VHa allotypes are discussed.

Amino Acid Sequence↗
PubMed1988

Transgenic rabbits with lymphocytic leukemia induced by the c-myc oncogene fused with the immunoglobulin heavy chain enhancer.

Transgenic rabbits with the rabbit c-myc oncogene fused with the rabbit immunoglobulin heavy chain enhancer region (E mu) DNA were developed by microinjecting pronuclei of single cell zygotes with the gene construct and implanting the microinjected eggs into pseudopregnant females. At age 17-20 days, 3 of 21 offspring born to seven females were found to have peripheral blood leukocyte counts of greater than 100,000 per mm3. Histology analyses showed extensive lymphocytic infiltration in the liver and kidney, indicating that these rabbits had a malignant lymphocytic leukemia. Genomic DNA analyses of thymus and peripheral blood lymphocytes revealed that the leukemic rabbits were transgenic and that both immunoglobulin heavy and kappa light chain genes were rearranged in the leukemic cells; thus, the leukemic cells are of B-cell lineage. One to four heavy and light chain gene rearrangements were observed, suggesting that the B-cell tumors were oligoclonal. Stable tissue culture cell lines from the bone marrow and peripheral blood of one of the transgenic rabbits have been developed. The development of B-cell leukemias in rabbits with the E mu-myc transgene contrasts with the development of B-cell lymphomas in mice carrying a similar transgene. The lymphomas in mice develop in adults and are monoclonal in origin. The leukemias in rabbits develop in juveniles, less than 3 weeks after birth, and appear oligoclonal in origin. The leukemias seem to develop in rabbit at a specific stage of development, and we suggest that a normal developmental signal may be involved in the oncogenesis.

Animals↗
PubMed1988

Conservation of an ATP-binding domain among RecA proteins from Proteus vulgaris, Erwinia carotovora, Shigella flexneri, and Escherichia coli K-12 and B/r.

The purified RecA proteins encoded by the cloned genes from Proteus vulgaris, Erwinia carotovora, Shigella flexneri, and Escherichia coli B/r were compared with the RecA protein from E. coli K-12. Each of the proteins hydrolyzed ATP in the presence of single-stranded DNA, and each was covalently modified with the photoaffinity ATP analog 8-azidoadenosine 5'-triphosphate (8N3ATP). Two-dimensional tryptic maps of the four heterologous RecA proteins demonstrated considerable structural conservation among these bacterial genera. Moreover, when the [alpha-32P]8N3ATP-modified proteins were digested with trypsin and analyzed by high-performance liquid chromatography, a single peak of radioactivity was detected in each of the digests and these peptides eluted identically with the tryptic peptide T31 of the E. coli K-12 RecA protein, which was the unique site of 8N3ATP photolabeling. Each of the heterologous recA genes hybridized to oligonucleotide probes derived from the ATP-binding domain sequence of the E. coli K-12 gene. These last results demonstrate that the ATP-binding domain of the RecA protein has been strongly conserved for greater than 10(7) years.

Adenosine Triphosphate↗
PubMed1988

Mortality of workers at acetylene production plants.

To reduce the risk of explosion oxyacetylene cylinders are filled with a spongy mass, acetone is added to saturate the mass, and acetylene is pumped into the cylinder. The first cylinders manufactured before 1936 used a kapok filling topped off with about 16 oz of crocidolite asbestos, with a metal gauze thimble inserted to reduce risk of flash back. Cylinders must be examined annually. The use of crocidolite ceased in 1972 and other fillings have been adopted since 1970; kapok cylinders now constitute less than 5% of the total stock. To assess possible hazards, a mortality study of workers first employed between 1935 and 1975 and followed up to December 1984 was undertaken. Simulation tests showed low concentrations of asbestos in the air even in the earliest period. The population studied consisted of 370 workers at the Bilston plant in the West Midlands, 611 at the 14 other plants in England and Wales, and 120 in Scotland. No deaths occurred from mesothelial tumours but there was an excess of deaths from cancer, particularly lung cancer, cancer of the stomach, and cancer of the pancreas, the latter accounting for eight deaths. Risks appeared to be concentrated at the Bilston plant. The importance of these findings is discussed.

Acetylene↗
PubMed1987

Rabbit IgA heavy chain genes: cloning and in vitro expression.

Screening of recombinant cosmid and phage libraries with a rabbit C alpha cDNA probe has identified a total of seven C alpha genes organized into two clusters; one cluster contains four genes and the other cluster, three genes. Southern blot analysis of genomic DNA indicates that the rabbit genome contains a minimum of ten C alpha genes; at least three genes remain to be cloned. Four of the seven cloned C alpha genes have been tested for their ability to be expressed in vitro. Each gene was cloned 3' to a rearranged murine VDJ gene, and the chimeric gene was then transfected into J558L plasmacytoma cells. Stable transfectants were selected, and each transfectoma was shown to express a chimeric alpha-heavy chain. These chimeric alpha-heavy chains were of two sizes, 55 kd and 60 kd. Each of the four transfectomas secreted chimeric heavy chains in association with endogenous murine lambda light chains. All four C alpha genes tested were expressible, indicating that the rabbit may have multiple IgA subclasses or isotypes. This is in marked contrast to mouse and human which have only one and two subclasses of IgA, respectively.

Animals↗
PubMed1987

Partial amino acid sequence analysis of rabbit MHC class II molecules isolated from two-dimensional polyacrylamide gels.

Partial N-terminal amino acid sequence analyses were performed on rabbit MHC class II molecules eluted from 2-D electrophoretic gels. Rabbit spleen cells were biosynthetically labelled with 3H-phenylalanine, 3H-tyrosine and 35S-methionine and class II molecules were immunoprecipitated with a monoclonal antibody, 2C4. The immunoprecipitates were electrophoresed on 2-D gels and as many as 15 spots were observed. Individual spots corresponding to alpha and beta chains were eluted from unfixed gels following visualization of the spots by autoradiography of 35S-Met labelled polypeptides. Ten eluted polypeptides were subjected to amino acid sequence analysis to locate Phe and Tyr residues. Comparison of these partial sequences with sequences of human class II molecules indicated that each of six beta chains and three of four alpha chains were homologous to human DQ molecules; one of the alpha chains appeared homologous to DR alpha or DP alpha. The assignment of alpha or beta chain to the polypeptides was confirmed by radiosequence of molecules labelled with 35S-Cys residues. Thus, by a relatively simple procedure, individual MHC class II polypeptides in spleen cell lysates have been separated from each other and partial amino acid sequences have been obtained.

Amino Acid Sequence↗
PubMed1987

Isolation of genes encoding bovine IgM, IgG, IgA and IgE chains.

Bovine C mu, C gamma, C alpha and C epsilon genes were cloned in an EMBL4 recombinant phage library using rabbit immunoglobulin switch mu (Su) and human C gamma as probes. Restriction mapping and Southern blot analyses of these clones identified one clone which hybridized with rabbit C mu and JH probes. The HG and C mu regions were separated by 6 kb of DNA. One C alpha and one C epsilon gene were found on overlapping clones and were separated by approximately 15 kb of DNA. Southern blot analysis of germline DNA with a bovine C alpha associated probe (S alpha) indicated that the germline contains a single C alpha gene. Similar analyses with a bovine C epsilon probe indicated that the germline contains either one C epsilon gene with allelic restriction polymorphism or two C epsilon genes. Three C gamma genes were cloned and did not overlap with one another. Southern blot analyses of germline DNA with a bovine C gamma probe indicated that the germline contains a total of four C gamma genes. The genes cloned correspond to three of the four genes identified by Southern blot analysis. The orientation of each CH gene was assigned by hybridization with S mu or S gamma probes. The S gamma probe hybridized to DNA immediately adjacent to all three C genes; the S probe hybridized to DNA immediately adjacent to the C mu, C alpha and C epsilon genes. Unexpectedly, the S mu probe also hybridized with a segment of DNA approximately 7 kb downstream of the C mu gene. This may represent a switch region for C gamma.

Animals↗
PubMed1987

Expression of rabbit IgA heavy chain genes in E. coli and in murine myeloma cells.

Rabbit IgA-heavy chain cDNA and germline genes were cloned into prokaryotic and eukaryotic expression vectors, respectively. The Fc alpha encoding portion of six C alpha cDNA clones were cloned into pUC8 and E. coli were transformed. Radioimmunoassay of the molecules synthesized by these clones showed that molecules with Fc alpha antigenic determinants were produced at the level of approximately 0.1 to 1.0 microgram per ml culture. Radiobinding analysis showed that each of the clones encoded heavy chains of the IgA-g subclass. Southern blot analysis of rabbit germline DNA revealed 10 germline C alpha genes. Five of these, isolated from recombinant cosmid libraries, were cloned into a eukaryotic expression vector containing a rearranged murine VDJ gene, the CH enhancer region and the Eco-gpt gene. Murine myeloma cells, J558L, were transfected with each of the heavy chain constructs and stable transfectants was selected with mycophenolic acid. The immunoglobulins produced by each transfectant were analyzed by radiobinding and by SDS-PAGE. Each transfectant were shown to synthesize IgA molecules and thus all five C alpha genes are expressible. The heavy chains from the transfectants ranged in size from 55,000 to 60,000 daltons. Radiobinding analyses indicated that four of the five genes encode molecules of the IgA-f subclass; the serological identity of the fifth gene is not yet established.

Animals↗
PubMed1986

Cytoplasmic immunofluorescence and light scatter analysis of lamina propria plasma cells by flow cytometry.

Cytoplasmic immunoglobulin of lamina propria plasma cells was analyzed by immunofluorescence on the flow cytometer. Lymphoid cells were made permeable to immunofluorescent reagents by treatment with Triton X-100. These cells were then reacted with FITC (green) anti-light chain and with phycoerythrin (red) anti-heavy chain antibodies. Flow cytometric analysis of these cells revealed that plasma cells expressing cytoplasmic immunoglobulin light and heavy chains were specifically stained by the immunofluorescent reagents. These plasma cells were also shown to have membrane Ig as detected by cell surface immunofluorescence. Light scatter analysis indicated that these plasma cells could be distinguished from lymphocytes by 90 degrees light scatter. These studies provide a method by which several parameters of gut plasma cells can be analyzed by flow cytometry.

Animals↗
PubMed1986

Rabbit major histocompatibility complex. I. Isolation and characterization of three subregions of class II genes.

Approximately 300 kb of DNA from the rabbit major histocompatibility complex class II region has been cloned from two genomic libraries. Four alpha chain genes and ten beta chain genes were identified in the recombinant phage and cosmid clones by hybridization with human class II cDNA probes. These genes were classified into three subregions (R-DP, R-DQ, and R-DR) based on hybridization analyses with human DP, DQ, and DR subregion genes under stringent conditions. Two alpha genes and one beta gene were assigned to the R-DP subregion, one alpha gene and one beta gene to the R-DQ subregion, and one alpha gene and eight beta genes to the R-DR subregion. In each subregion, the alpha gene and at least one beta gene were closely linked and were oriented in a manner similar to those in the homologous subregions of human and mouse. A combination of cloning data and genomic blot analyses indicated that the rabbit genome contains a minimum of four alpha-chain genes. The results suggested that there has been evolutionary conservation of the subunit organization of the alpha- and beta-chain genes as well as the coding sequences of these genes and that the mammalian ancestor possessed three distinct MHC class II subregions before diversification of human, mouse, and rabbit.

Animals↗
PubMed1986

Nucleotide binding by a 24-residue peptide from the RecA protein of Escherichia coli.

We have recently demonstrated that two ATP analog affinity labels, 8-azidoadenosine 5'-triphosphate (N3ATP) and 5'-p-fluorosulfonylbenzoyladenosine (5'FSBA), covalently modify RecA protein of Escherichia coli at a specific tyrosine residue (Tyr-264) located within a 24-residue tryptic peptide (T-31) spanning residues 257-280. Here we show that N3ATP efficiently modifies purified peptide T-31 and show that the interaction is specific by the following criteria: photolabeling of peptide T-31 is saturable with respect to the N3ATP concentration; photolabeling is competitive with ATP and adenosine but not with adenine, UTP, or TTP; and other peptides derived from RecA protein were poor substrates for photolabeling except for one fragment that showed a nonspecific interaction with the photoaffinity analog. Analysis of N3ATP-modified T-31 shows that the photolabel attaches to more than one site within the peptide. These data argue that peptide T-31 contains some sites of contact for adenine and ribose moieties of ATP when it is bound to RecA protein.

Adenosine Triphosphate↗