3'-minor groove binder-DNA probes increase sequence specificity at PCR extension temperatures.

DNA probes with conjugated minor groove binder (MGB) groups form extremely stable duplexes with single-stranded DNA targets, allowing shorter probes to be used for hybridization based assays. In this paper, sequence specificity of 3'-MGB probes was explored. In comparison with unmodified DNA, MGB probes had higher melting temperature (T(m)) and increased specificity, especially when a mismatch was in the MGB region of the duplex. To exploit these properties, fluorogenic MGB probes were prepared and investigated in the 5'-nuclease PCR assay (real-time PCR assay, TaqMan assay). A 12mer MGB probe had the same T(m)(65 degrees C) as a no-MGB 27mer probe. The fluorogenic MGB probes were more specific for single base mismatches and fluorescence quenching was more efficient, giving increased sensitivity. A/T rich duplexes were stabilized more than G/C rich duplexes, thereby leveling probe T(m)and simplifying design. In summary, MGB probes were more sequence specific than standard DNA probes, especially for single base mismatches at elevated hybridization temperatures.

Triplex targeting of a native gene in permeabilized intact cells: covalent modification of the gene for the chemokine receptor CCR5.

A 12 nucleotide oligodeoxyribopurine tract in the gene for the chemokine receptor CCR5 has been targeted and covalently modified in intact cells by a 12mer triplex forming oligonucleotide (TFO) bearing a reactive group. A nitrogen mustard placed on the 5'-end of the purine motif TFO modified a guanine on the DNA target with high efficiency and selectivity. A new use of a guanine analog in these TFOs significantly enhanced triplex formation and efficiency of modification, as did the use of the triplex-stabilizing intercalator coralyne. This site-directed modification of a native chromosomal gene in intact human cells under conditions where many limitations of triplex formation have been partially addressed underscores the potential of this approach for gene control via site-directed mutagenesis.

Sequence-specific targeting and covalent modification of human genomic DNA.

We compare two techniques which enable selective, nucleotide-specific covalent modification of human genomic DNA, as assayed by quantitative ligation- mediated PCR. In the first, a purine motif triplex-forming oligonucleotide with a terminally appended chlorambucil was shown to label a target guanine residue adjacent to its binding site in 80% efficiency at 0.5 microM. Efficiency was higher in the presence of the triplex-stabilizing intercalator coralyne. In the second method, an oligonucleotide targeting a site containing all four bases and bearing chlorambucil on an interior base was shown to efficiently react with a specific nucleotide in the target sequence. The targeted sequence in these cases was in the DQbeta1*0302 allele of the MHC II locus.

Structure of mink immunoglobulin gamma chain cDNA.

Two cDNA clones, encoding mink Ig gamma chains were characterized. The pIGG47 clone contains a part of the leader segment, VDJ and C regions, and pIGG14 contains a part of the J and a complete C region. The clones differ by only four nucleotides in the C region, and they most probably represent allelic variants of the same gene. The V gene segment of pIGG47 was found to be highly similar to human VHIII subgroup sequences; there was 86-87% similarity for the whole V gene segment and 91% for the VHIII specific regions (codons 65-87). Southern blot analysis demonstrated that a high proportion of mink VH genes is VHIII related. The V gene segment used as a probe revealed 19-23 bands in mink DNA under stringent conditions. This is in agreement with our previous data showing that a high proportion of mink Ig contains an 'alternative' binding site for protein A, a feature common to VHIII-related molecules. According to Southern blot analysis there may be 5-7 C gamma genes at the mink IgH locus.

Chromosomal and regional localization of the loci for IGKC, IGGC, ALDB, HOXB, GPT, and PRNP in the American mink (Mustela vison): comparisons with human and mouse.

Chromosomal localization of the genes for gamma- and kappa-immunoglobulins (IGGC and IGKC, respectively), aldolase B (ALDB), prion protein (PRNP), homeo box B (HOXB), and glutamate pyruvate transaminase (GPT) were determined with the use of mink-rodent hybrid cells. Analysis of segregation of the mink markers and chromosomes in these hybrid cells allowed us to assign the gene for HOXB to Chromosome (Chr) 8, IGGC to Chr 10, PRNP and IGKC to Chr 11, ALDB to Chr 12, and GPT to Chr 14 in mink. Furthermore, using a set of mink-mouse hybrid cells carrying fragments of mink Chr 8 of different sizes, we assigned the gene for HOXB to the pter-p26 region of the short arm of Chr 8. Comparative mapping of the genes of mink, human, and mouse, as well as other mammalian species, demonstrated that the mink genes HOXB, PRNP, ALDB, and IGGC are members of a conserved region shared by many mammalian species in common; the IGKC gene is a member of a conserved region common to carnivores and primates, not rodents; the GPT gene is a member of a syntenic gene group probably unique to the Mustelidae family or carnivores.

Expression of immunoglobulin kappa and lambda chains in mink.

The ratio of kappa and lambda chains of immunoglobulins varies significantly from one species to another. It has previously been thought that lambda was only type expressed in mink. We tested mink immunoglobulin light chains using two monoclonal antibodies G80 and G88. It has been shown that G80 and G88 specifically recognize two antigenically different subpopulations of the light chains. Immunochemical analysis of these subpopulations separated by affinity chromatography suggested that they represent lambda and kappa types of light chains, respectively. Screening of a mink cDNA library with monoclonal antibody G88 resulted in the isolation of clone pIGK-1 containing kappa chain-encoding sequence. The cDNA insert of pIGK-1 included most of the V segment, as well as the J, C and 3' untranslated sequences. Mink V kappa sequence shown the highest homology with the human V kappa II subgroup genes (76-79%). Mink C kappa sequence was 53-63% homologous to C kappa of other species. The striking feature of mink C kappa chain is the presence of glutamine in the C-terminal position. Southern blot analysis suggested that mink haploid genome has one C kappa gene and multiple V kappa genes. The kappa:lambda chain ratio in the 12 minks studied was, on the average, 46:54. The same ratio was observed for the kappa- and lambda-producing cells in the mesenteric lymph nodes. The five previously identified mink light chain allotypes were assigned to the lambda chains, thereby confirming that lambda chains in this species are additionally subdivided into several subtypes.

The mink gene for the lambda light immunoglobulin chain: characterization of cDNA and chromosomal localization.

A cDNA library from mink spleen was constructed by use of the phage lambda gt11. The library was screened using polyvalent serum raised against the mink immunoglobulin lambda chain. As a result, several clones expressing mink immunoglobulin lambda light chains were identified. Sequencing of one of the clones with an 803 bp insert was performed. The insert comprised nearly the entire coding region for the mature lambda light immunoglobulin gene with the exception of the leader polypeptide and several amino acids of the FR1 region of the V segment. Compared with the rabbit, mouse and human lambda light immunoglobulin genes, the homology of the cloned sequence was found to be highest relative to the rabbit gene. With the cloned mink cDNA containing the C-region only as a probe, the DNAs from mink-Chinese hamster hybrid clones were studied. The results of segregation analysis of this mink cDNA sequence and mink chromosomes in the mink-Chinese hamster clone panel allowed us to assign the gene for the lambda light immunoglobulin constant polypeptide (IGLC) to mink Chromosome (Chr) 4.

[The kinetics of specific serum antibodies in sheep infested with sheep bot larvae]. / Kinetika spetsificheskikh syvorotochnykh antitel u ovets, invazirovannykh lichinikami ovech'ego ovoda.

The kinetics of specific antibodies of the blood serum of sheep experimentally infested with 80, 160 and 1000 specimens of Oestrus ovis larvae was examined. The affinity pure serum IgG and the immunoferment analysis (ELISA) were used for qualitative estimation of specific antibodies. It has been shown that the level of specific antibodies correlates with the larval biomass and is connected with ontogenesis of this parasite. The younger animals, which were infested for the first time, are characterized by more intensive production of specific IgG than adult reinfested ones. The ways of immunity response formation in animals infested with Oestrus ovis larvae are considered.

[Cloning and sequencing of immunoglobin lambda-chain cDNA in american mink (Mustela vison]. / Klonirovanie i analiz pervichnoi struktury kDNK lambda-tsepi immunoglobulinov amerikanskoi norki (Mustela vison).

cDNA library in the lambda gt11 phage was constructed using poly (A+)-mRNA from mink spleen as a template. Immunoscreening of the library allowed the identification of 2 lambda-related clones containing 370 and 803 bp insertion (lambda IGL-1 and lambda IGL-2). Analysis of the primary structure of lambda IGL-2 demonstrated that it contains a large portion of V lambda-segment, J lambda-segment, C lambda-gene and its 3'-untranslated part. The nucleotide sequences known for the immunoglobulin genes were compared to the sequence of the lambda IGL-2 clone. The highest degree of homology was established for the rabbit lambda-genes, this being 63, 94 and 72% for the RF3 region of V lambda-segment, J lambda-segment and C lambda-region, respectively.

Genetic polymorphism of IgG in the mink. V. Two new genetic markers of the lambda light chains of mink immunoglobulins, L4 and L5.

Two new allotypes of the light (L) chains IgG, L4 and L5, were identified in the mink with dispecific antiserum produced by immunization with allogenic IgG. By means of hybrid IgG molecules and proteolytic fragments, L4 and L5 were localized on the C region of the L chain. L4 and L5 occurred frequently in the three mink populations studied and L4 and L5 are inherited independently of the known mink C gamma allotypes. L4 and L5 are encoded by closely linked genes. The antigenic specificities of L4 and L5 were not identified in the closely related Mustelidae and in the other mammalian representatives. Consequently, L4 and L5 are species specific to mink. Determination of the phenotype combinations of the five allotypes on the L chains (including the new L4 and L5) demonstrated the existence of seven combinations only with a predominance of L1,2,3; L4,5, and L1,2,3,4,5 phenotypes. Based on the results obtained, it is concluded that the mink C lambda locus has a complex organization. A model for the mink C lambda locus with at least three or possibly five linked genes is suggested.

Mink-mouse hybridomas that secrete mink immunoglobulin G.

Optimum conditions were established to obtain mink-mouse interspecific hybridomas secreting mink IgG in fusions of mouse myelomas with mink immune spleen cells. Minks were immunized with allogeneic IgG, and the spleen cells were fused with three mouse myeloma lines P3-X63-Ag8.653, NSO and Sp2/0-Ag14. Of these, P3-X63-Ag8.653 and NSO were found to be the best fusion partners giving the highest yield of hybrid clones and number of IgG secreting clones. Cloning of mink-mouse hybridomas was efficient when BALB/c nu/nu peritoneal and spleen cells were used as feeders. The ten clonal lines produced secreted intact mink IgG molecules as shown by SDS-PAGE and subsequent immunoblotting. The secretion level of IgG ranged from 5 to 200 ng/ml in the clonal lines.