Expression and sequence analysis of a cDNA encoding the orotidine-5'-monophosphate decarboxylase domain from Ehrlich ascites uridylate synthase

Orotidine-5'-monophosphate decarboxylase (OD-Case) catalyzes the conversion of orotidine 5'-monophosphate to UMP. In mammals, ODCase is present as part of a bifunctional protein which also contains orotate phosphoribosyltransferase; the preceding enzyme in the de novo UMP biosynthetic pathway. We have isolated a plasmid (pMEJ) which contains a cDNA for the ODCase domain of UMP synthase. Insertion of this sequence into an Escherichia coli expression vector (pUC12) has allowed for the expression of ODCase and not orotate phosphoribosyltransferase in E. coli. The molecular weight of the expressed protein is 26,000-27,300 from immunoblot analysis which corresponds closely to the molecular weight of the ODCase domain (28,500) isolated by tryptic digestion of UMP synthase. We have sequenced the cDNA insert of pMEJ and deduced the amino acid sequence. The molecular weight of the ODCase domain calculated from the amino acid sequence in 28,654. Comparison of the deduced amino acid sequence from pMEJ with that for yeast ODCase (a monofunctional protein) demonstrated that 52% of the amino acids were identical when the two sequences are compared. Furthermore, several stretches of the amino acid sequence have 80% or greater absolute homology.     

Protein engineering of homodimeric tyrosyl-tRNA synthetase to produce active heterodimers

Heterodimers of tyrosyl-tRNA synthetase from Bacillus stearothermophilus have been produced by mutagenesis at the subunit interface. Oppositely charged groups have been engineered into the subunits so that they can form a complementary pair. Wild-type tyrosyl-tRNA synthetase is a symmetrical dimer in which the side chains of the 2 Phe-164 residues interact at the subunit interface. Phe-164 was mutated to Asp in tyrosyl-tRNA synthetase and to Lys in a truncated enzyme (des-(321-419)tyrosyl-tRNA synthetase) which lacks the two tRNA-binding sites, but which can catalyze pyrophosphate exchange. The size difference allows subunit association to be studied by gel filtration chromatography. These changes induce reversible dissociation from active dimers into inactive monomers at pH values which favor ionization at position 164. A mixture of the two mutants near neutral pH is apparently fully active in pyrophosphate exchange and consists of a heterodimer of [Asp164]tyrosyl-tRNA synthetase and [Lys164]des-(321-419)tyrosyl-tRNA synthetase. Despite having only one binding site for tRNA, heterodimer has full aminoacylation activity at high concentrations of tyrosine. We have therefore produced a family of dimers that differ in stability near neutral pH. This novel approach using protein engineering allows specific dimerization of subunits of the same size that have different defined mutations, each subunit being tagged by the charge. Such hybrid proteins can be used to study subunit interaction.     

Imino 1H- and 31P-NMR analysis of the interaction of propidium and ethidium with DNA

At low temperature and low salt concentration, both imino proton and ³¹p-nmr spectra of DNA complexes with the intercalators ethidium and propidium are in the slow-exchange region. Increasing temperature and/or increasing salt concentration results in an increase in the site exchange rate. Ring-current effects from the intercalated phenanthridinium ring of ethidium and propidium cause upfield shifts of the imino protons of A · T and G · C base pairs, which are quite similar for the two intercalators. The limiting induced chemical shifts for propidium and ethidium at saturation of DNA binding sites are approximately 0.9 ppm for A · T and 1.1 ppm for G · C base pairs. The similarity of the shifts for ethidium and propidium, in both the slow- and fast-exchange regions over the entire titration of DNA, shows that a binding model for propidium with neighbor-exclusion binding and negative ligand cooperativity is correct. The fact that a unique chemical shift is obtained for imino protons at intercalated sites over the entire titration and that no unshifted imino proton peaks remain at saturation binding of ethidium and propidium supports a neighbor-exclusion binding model with intercalators bound at alternating sites rather than in clusters on the double helix. Addition of ethidium and propidium to DNA results in downfield shifts in ³¹P-nmr spectra. At saturation ratios of intercalator to DNA base pairs in the titration, a downfield shoulder (approximately ?2.7 ppm) is apparent, which accounts for approximately 15% of the spectral area. The main peak is at ?3.9 to ?4.0 ppm relative to ?4.35 in uncomplexed DNA. The simplest neighbor-binding model predicts a downfield peak with approximately 50% of the spectral area and an upfield peak, near the chemical shift for uncomplexed DNA, with 50% of the area. This is definitely not the case with these intercalators. The observed chemical shifts and areas for the DNA complexes can be explained by models, for example, that involve spreading the intercalation-induced unwinding of the double helix over several base pairs and/or a DNA sequence- and conformation-dependent heterogeneity in intercalation-induced chemical shifts and resulting exchange rates.     

Autoradiographic detection of HPRT variants of human lymphocytes resistant to RNA synthesis inhibition

The feasibility of using RNA synthesis in freshly isolated, human peripheral blood lymphocytes to detect 6-thioguanine (TG)- and 8-azaguanine (AG)-resistant variants in an autoradiographic assay similar to that of Strauss and Albertini (1979) has been evaluated. In phytohemagglutinin (PHA)-stimulated cultures RNA synthesis and HPRT activity began well in advance of DNA synthesis and increased in parallel during the first 44 h of culture. Introduction of TG or AG with PHA at the beginning of culture completely inhibited DNA synthesis during the first 44 h and reduced RNA synthesis to low levels within 24 h. When TG or AG was added after cells had been in culture for 38 h, DNA synthesis was reduced quickly while RNA synthesis was inhibited more slowly. An autoradiographic assay is described in which freshly isolated lymphocytes are cultured with PHA for 24 h, with or without TG or AG, then labeled with [3H]uridine for 1 h. TG-resistant and AG-resistant variant frequencies for 2 normal individuals and a Lesch-Nyhan individual were determined with this assay. The variant frequencies for the normal individuals ranged from 0.46 to 10.6 X 10(-5) depending upon the selective conditions used. All the Lesch-Nyhan cells were resistant to 0.2 microM-2 mM AG; some were sensitive to 0.2 mM TG and most were sensitive to 2.0 mM TG.     

Amino acid sequence homologies between rabbit, rat, and human serum retinol-binding proteins

The main transporting protein for vitamin A in rabbit serum, the retinol-binding protein (RBP), was isolated and its amino acid sequence determined. Rabbit RBP was found to be highly homologous to human RBP, whose amino acid sequence was elucidated earlier, and to rat RBP. The rat RBP sequence was obtained by combining information deduced from the nucleotide sequences of two overlapping cDNA clones with the NH2-terminal sequence of the isolated protein determined by automated Edman degradation. The identity between the three proteins is approximately 90%. The high degree of homology between RBP molecules from different species is probably explained by the fact that RBP participates in at least three types of molecular interactions: in the binding of prealbumin, in the interaction with retinol, and in the recognition of a specific cell surface receptor. All these interactions should lead to a conservation of RBP structure. The amino acid differences between rabbit, rat, and human RBP are discussed in light of the recent elucidation of the three-dimensional structure of human RBP. Hybridization of a probe isolated from a rat RBP cDNA clone to restriction enzyme-digested genomic DNA from rat and mouse suggests that RBP is encoded by a single gene.     

Production of (S)-3-chlorolactaldehyde from (S)-alpha-chlorohydrin by boar spermatozoa and the inhibition of glyceraldehyde 3-phosphate dehydrogenase in vitro

The (S)-isomer of the male antifertility agent alpha-chlorohydrin was metabolized by mature boar spermatozoa in vitro to (S)-3-chlorolactaldehyde. This oxidative process, which did not occur when (R)-alpha-chlorohydrin was offered as a substrate, was catalysed by an NADP+-dependent dehydrogenase that converts glycerol to glyceraldehyde. (S)-3-chlorolactaldehyde, produced by this metabolic reaction or when added to suspensions of boar spermatozoa, was a specific inhibitor of glyceraldehyde 3-phosphate dehydrogenase as assessed by the accumulation of fructose 1,6-bisphosphate and the triosephosphates. When glycerol and (S)-alpha-chlorohydrin were added concomitantly to boar spermatozoa in vitro, the presence of glycerol decreased the degree of inhibition of glyceraldehyde 3-phosphate dehydrogenase. Extracts of glyceraldehyde 3-phosphate dehydrogenase that were obtained from boar spermatozoa incubated with (S)-alpha-chlorohydrin or (R,S)-3-chlorolactaldehyde showed significant reductions in their enzymic activity.     

Variation in natural killer activity in peripheral blood during the menstrual cycle.

Natural killer activity was measured sequentially in normal female volunteers through their menstrual cycle. During the periovulatory period there was a significant fall in natural killer activity compared with in normal male volunteers. This variation was not apparent in women taking oral contraception. Cytotoxic activity was not related to oestradiol concentrations in individual women. The data support an interaction between immunological activity and sex hormones over the normal physiological range and would account for the described reduction in natural killer activity in pooled blood from female blood donors.