Structural studies and isolation of cDNA clones providing the complete sequence of rat liver dihydropteridine reductase

The cleavage of reductively alkylated rat liver dihydropteridine reductase with cyanogen bromide afforded a mixture of peptides, six of which (CB-1 to CB-6) were isolated and purified by C8 reverse-phase high performance liquid chromatography. Portions of peptides CB-1, CB-4, and CB-6 were sequenced by automated Edman degradation and high performance liquid chromatography and the carboxyl-terminal region by conventional procedures. Further proteolytic digestion of CB-6 and isolation of the products afforded a seven-amino acid peptide. A low degeneracy probe comprising 20 nucleotides was synthesized from the sequence of this peptide and was used to screen a rat liver cDNA expression library constructed in the vector lambda gt 10. Positive clones were isolated, and detailed examination of five of these by restriction endonucleases and dideoxy sequence analyses allowed identification of the entire coding region for dihydropteridine reductase. The gene was found to code for a protein of 240 amino acids (excluding the methionine initiator) of Mr = 25,420. Each of the sequences corresponding to the peptides CB-1, CB-4, CB-6, and the carboxyl terminus were identified in the deduced protein sequence. The rat enzyme is highly homologous to the human dihydropteridine reductase; the two proteins differ in only 10 amino acids, and all are conservative substitutions. In contrast, the sequence shows little homology with that of mammalian dihydrofolate reductase: reduced pyridine nucleotide-requiring enzymes with superficial mechanistic similarities.     

Receptor diversity of insulin-specific T cell lines from C57BL (H-2b) mice

To characterize the T cell receptor repertoire in an immune response in which the Ia and nominal antigenic determinants are defined and limited, we have cloned and sequenced the expressed receptors from four independent, beef insulin-specific T cell lines from C57BL mice. Each of these lines responded to beef but not to the pork insulin, thus defining the nominal antigenic determinant recognized. Furthermore, each of these lines could only be presented antigen by B6 but not mutant B6.C-H-2bm12 antigen-presenting cells, thus defining the requisite Ia recognition or antigen-association site. In spite of this functional similarity in ligand specificity, each of these T cell lines was found to use different V alpha and V beta gene segments. Moreover, structural comparisons of implied protein sequences of each of these receptors showed no stretches of conserved amino acid residues that could be implicated in ligand interaction. However, the V alpha genes used by these four clones appeared considerably more homologous to each other than were their V beta genes.     

Synthesis and efficient isolation procedure for gamma-linked fluorescein methotrexate

Fluorescein isothiocyanate was reacted in dimethyl sulfoxide with a ten-fold excess of diaminopentane, and the mono-substituted thiourea product was isolated by DEAE-cellulose chromatography, lyophilization and acid precipitation from aqueous base. The dried product was then condensed in dry dimethyl sulfoxide with Methotrexate (MTX) activated by prior incubation (30 min) with 1-ethyl-3-(3'-dimethylaminopropyl) carbodiimide hydrochloride, and the reaction products were purified by column chromatography on DEAE-cellulose. Exhaustive elution with 1 M ammonium bicarbonate removed several by-products then finally afforded the exclusively gamma-linked fluorescein--MTX derivative. After lyophilization and acid-base precipitation the compound was obtained in good yield (greater than 40%), was homogeneous by reverse-phase HPLC epsilon 493 (0.1 N NaOH) = 66,000 and was a comparable inhibitor to MTX for rat-liver dihydrofolate reductase.     

DNA-RNA hybridization studies of gene activity during the development of the gastropod, Acmaea scutum

DNA-RNA molecular hybridization experiments were performed to compare sequences of RNA that are present in the unfertilized egg, gastrula, trochophore, early veliger, mid-veliger, and adult with that population that is being synthesized by the midveliger. The conditions of annealing were designed to examine the more common RNA sequences that are complementary to the highly reiterated sequences of the genome. It was found that the RNA of the unfertilized egg competes extensively with RNA sequences that are being synthesized by this relatively late larval stage. These RNA sequences are present at a lower concentration in RNA preparations extracted from unfertilized eggs as compared to later stages. Autoradiographs of ³H-uridine labeled veligers indicate that the pulse-labeled RNA used in these experiments represents that synthesized by a wide variety of larval tissues. These results are discussed with respect to differences in RNA metabolism and determination between regulative and mosaic embryos.