A procedure is described for the isolation of highly purified heavy-chain immunoglobulin mRNAs from a variety of mouse plasmacytomas (IgA, IgG, and IgM producers). The use of fresh tissue and the rapid isolation and direct extraction of membrane-bound polyribosomes were found to be essential in obtaining large quantities of undegraded heavy-chain mRNAs. The individual mRNAs were purified by two cycles of oligo(dT)-cellulose chromatography, sodium dodecyl sulfate--sucrose gradient centrifugation, and electrophoresis on 98% formamide containing polyacrylamide gels. When added to a cell-free protein-synthesizing system from wheat germ, the MPC-11 gamma2b and H2020 alpha heavy-chain mRNAs efficiently directed the synthesis of a predominant product of 55 000 molecular weight, while the synthesis of a 70 000 dalton protein in addition to other lower molecular weight polypeptides were observed with MOPC 3741 mu mRNA. All of these proteins were immunoprecipitable with class-specific heavy-chain antisera, and in the case of the gamma2b in vitro products good correspondence in a comparative trypsin--chymotrypsin fingerpring with in vivo labeled gamma2b heavy chain was observed. The gamma2b and a alpha heavy-chain mRNAs possessed a chain length of approximately 1800 nucleotides and the mu mRNA a size of approximately 2150 nucleotides when examined under stringent denaturation conditions. The purities of the alpha, gamma2b, and mu mRNAs were estimated to be 60--80%, 50--70%, and 50--83%, respectively, on the basis of their hybridization rates with cDNA probes in comparison to mRNA standards of known complexity. Heavy-chain mRNAs of the same class isolated from different mouse strains (Balb/C or NZB) display no detectable sequence differences in cross hybridization experiments, even though the cDNA--mRNA hybrids are submitted to stringent S1 nuclease digestion. These results indicate that allotypic determinants represent only a minor fraction of the heavy-chain constant region sequence in the mouse.

The number of germline genes specifying the variable sequences of the Vkappa21 group of light chains was determined by saturation hybridisation analysis to be four to six. This gene multiplicity is considerably less than the total variability in the group, indicating that kappa-chain diversity is generated by somatic mutations in both framework and complementarity-determining (hypervariable) regions.

Environmental conditions affect biocontrol agents in a field, being appropriate formulations an alternative to overcome this problem. Formulations based on Trichoderma asperellum TV190 were prepared by emulsified mineral or vegetable oils, which protected spores from ultraviolet radiation, showing greater viability of 37-43% (mineral) and 56-63% (vegetable) than the control (8-12%). These formulations improved an antagonism of T. asperellum on Rhizoctonia solani under greenhouse conditions, reducing infected corn seedlings by 72% (mineral) and 59% (vegetable). Necrotic spot size was reduced by 90.04% (mineral) and 87.29% (vegetable). A granular formulation, prepared with degreased corn germ and T. asperellum spores, protected the corn seedlings from R. solani under greenhouse conditions, with 73% reduction of infected plants and 93% reduction of necrotic spot size. Both granular and liquid formulations were able to improve T. asperellum antagonism, suggesting that these formulations could be included in agricultural pest control strategies.

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The A comparison of karyotypic data with hybridization data has limited the possible locations of the Ig genes to only a few chromosomes.In the mouse, as in other mammals, the chromosomal locations of the genes coding for antibody heavy and light chain structures have not been clearly identified. From genetic studies it has been inferred that these genes are located on different autosomes (1), and it has been suggested on the basis of a variable region marker that the mouse K locus may be on chromosome 6 (2). However, owing to the lack of a demonstrable linkage between heavy or light chain constant-region genes and other loci and to the possible existence of regulatory loci affecting the expression of the immunoglobulin structural genes (3, 4), a more definitive study is called for. We have therefore attempted to solve this problem by use of the molecular hybridization techniques. In these experiments, cDNA probes representing the constant regions of mouse K and AI light chain mRNAs and a, 72h and M heavy chain mRNAs were annealed to a large excess of DNA from a series of mouse-human hybrid cell lines that are deficient for various mouse chromosomes (5). A similar approach using hybrids segregating human chromosomes has shown that the structural gene for human a-globin is on chromosome 16 (6). The results of our studies demonstrate clearly that genes CA, CK, and CH are autosomal and not linked. Moreover, a comparison of karyotypic and hybridization data has limited the possible locations of these genes to only a few chromosomes. Interestingly, our results indicate that the chromosome containing the CK gene is probably not chromosome 6. 55-14, 55-54, and 55-91 were derived from three independent fusions of HT-1080-6TG human fibrosarcoma cells with BALB/c mouse peritoneal macrophages; the hybrid line 55-84 was derived from a fusion of HT-1080-6TG cells with OTT6050 mouse teratocarcinoma cells (5). The F numbers refer to different flasks in which hybrids were formed following the initial fusion event, and Cl numbers refer to different clonal isolates from the initial hybrid cell cultures. Line VII is an early sample of the 55-14 F7 culture that was kept frozen until its cultivation for the present series of experiments. Karyotype analyses were carried out by using the trypsin-Giemsa banding technique (5). MATERIALS AND METHODSDNAcDNA Annealing Experiments. The cDNA probes were synthesized from highly purified heavy and light chain mRNAs isolated from various mouse plasmacytomas. The tumors used as sources of the particular mRNAs were: AI, H2020