Saltatory forward movement of a poly(A) polymerase during poly(A) tail addition

Vaccinia poly(A) polymerase (VP55) interacts with > or = 33-nucleotide (nt) primers via uridylates at two sites (-27/-26 and -10). It adds approximately 30-nt poly(A) tails with a rapid, processive burst in which the first few nt are added without substantial primer movement, and addition of the remaining adenylates is dependent upon a six-uridylate tract at the extreme 3' end of the primer and accompanied by polymerase translocation. Interaction of VP55 with 2-aminopurine (2-AP)-containing primers was associated with a 3-fold enhancement in 2-AP fluorescence. In stopped-flow experiments, fluorescence intensity changed with time during the polyadenylation burst in a manner dependent upon the position of 2-AP, indicating a non-uniform isomerization of the polymerase-primer complex with time consistent with a discontinuous (saltatory) translocation mechanism. Three distinct translocatory phases could be discerned: a -10(U)-binding site forward movement, a -27/-26(UU)-binding site jump to -10, then a -27/-26(UU)-binding site movement further downstream. Poly(A) tail elongation showed no apparent pauses during these isomerizations. Fluorescence changes during polyadenylation of 2-AP-containing primers with short preformed oligo(A) tails reinforced the above observations. Primers composed entirely of oligo(U) (apart from the 2-AP sensor), in which the polymerase modules might be most able to "slide" uniformly, also showed the characteristic saltatory pattern of translocation. These data indicate, for the first time, a discontinuous mode of translocation for a non-templated polymerase.     

A dominant (mutD5) and a recessive (dnaQ49) mutator of Escherichia coli

The two known strong mutators of Escherichia coli K12, mutD5 (Degnen & Cox, 1974) and dnaQ49 (Horiuchi et al., 1978), are located at almost the same position, at five minutes on the linkage map. To clarify the genetical and functional relationships between these two mutators, we have constructed hybrid plasmids and phages carrying dnaQ+ or mutD5 by using in vivo and in vitro recombination techniques and examined their effect on the phenotype of wild-type or mutant bacteria. The results indicated that the mutD5 mutator is dominant over the wild-type allele whereas dnaQ49 is recessive. Thus, mutD5 plasmid or mutD5 transducing lambda phage can be used to convert a wild-type strain to a highly mutable strain. Both dnaQ+ and mutD5 plasmids carried a 1.5 X 10(3) base DNA fragment derived from the E. coli chromosome and they were indistinguishable from each other by restriction enzyme analysis. Moreover, specific labeling of the plasmid-encoded proteins by the maxicell method revealed that the mutD5 plasmid codes for two proteins, one whose molecular weight is 25,000 and the other whose molecular weight is 21,000, which correspond to the dnaQ protein and RNase H, respectively. Insertion of the gamma delta sequence into the mutD gene of the plasmid resulted in disappearance of the 25,000 Mr protein. These results suggested that the dnaQ49 and mutD5 mutator are mutations that have arisen in a single gene, though they differ in many respects.     

A ligand-induced conformational change in apolipoprotein(a) enhances covalent Lp(a) formation

Lipoprotein(a) (Lp(a)) assembly proceeds via a two-step mechanism in which initial non-covalent interactions between apolipoprotein(a) (apo(a)) and low density lipoprotein precede disulfide bond formation. In this study, we used analytical ultracentrifugation, differential scanning calorimetry, and intrinsic fluorescence to demonstrate that in the presence of the lysine analog epsilon-aminocaproic acid, apo(a) undergoes a substantial conformational change from a "closed" to an "open" structure that is characterized by an increase in the hydrodynamic radius (approximately 10%), an alteration in domain stability, as well as a decrease in tryptophan fluorescence. Although epsilon-aminocaproic acid is a well characterized inhibitor of the non-covalent interaction between apo(a) and low density lipoprotein, we report the novel observation that this ligand at low concentrations (100 microm-1 mm) significantly enhances covalent Lp(a) assembly by altering the conformation of apo(a). We developed a model for the kinetics of Lp(a) assembly that incorporates the conformational change as a determinant of the efficiency of the process; this model quantitatively explains our experimental observations. Interestingly, an analogous conformational change has been previously described for plasminogen resulting in an increase in the hydrodynamic radius, an increase in tryptophan fluorescence, and an acceleration of the rate of plasminogen activation. Although the functions of apo(a) and plasminogen have diverged considerably, elements of structural and conformational homology have been retained leading to similar regulation of two unrelated biological processes.     

Quantitation of Gibberellins A(1), A(3), A(4), A(9) and a Putative A(9)-Conjugate in Grafts of Sitka Spruce (Picea sitchensis) during the Period of Shoot Elongation

The levels of endogenous gibberellin A₁ (GA₁), GA₃, GA₄, GA₉, and a cellulase hydrolyzable GA₉ conjugate in needles and shoot stems of mature grafts of Sitka spruce (Picea sitchensis [Bong.] Carr.) grown under environmental conditions that were either inductive, hot, and dry, or noninductive, cool, and wet, for flowering, were estimated by combined gas chromatography-mass spectrometry selected ion monitoring using deuterated [²H₂]GA₁, GA₃, GA₄, and GA₉ as internal standards. The samples were taken when the shoots had elongated about 30, 70, and 95% of the final shoot length and 17 days after elongation had terminated. The concentration of putative GA₉-conjugate, estimated by GCSIM of GA₉ after cellulase hydrolysis of the highly water soluble fraction, was 33 nanograms per gram fresh weight in the needles of both heat and drought- and cool and wet-treated plants sampled just after bud burst. The concentration gradually decreased to a final value of 13 nanograms per gram fresh weight in the heat and drought-treated grafts and 6 nanograms per gram fresh weight in the cool and wet-treated grafts. The stems contained no detectable putative GA₉ conjugate. Free GA₉ was highest in heat and drought-treated material. For plants subjected to this treatment, GA₉ increased from 22 to 32 nanograms per gram fresh weight in needles and from 1 to 22 nanograms per gram fresh weight in stems during the rapid stem elongation phase. By day 17, after cessation of shoot elongation, GA₉ had decreased to 12 nanograms per gram fresh weight in needles and 9 nanograms per gram fresh weight in the shoot stems. The cool and wet-treated material also showed an increase in GA₉ concentration during shoot elongation. However, the concentration was not as high and was also delayed compared with heat and drought-treated material. By day 17, after cessation of shoot elongation, GA₉ concentration was 9 nanograms per gram fresh weight in needles and 5 nanograms per gram fresh weight in stems for cool and wet treatment plants. The concentration of GA₄ was very low in tissue from both treatments. Fluctuation in concentration of the more polar gibberellins, GA₁ and GA₃, showed the same pattern as fluctuations in the content of GA₉. However, the heat and drought-treated material had lower amounts of GA₁ and GA₃ during the later phases of shoot elongation, than the cool and wet-treated material. These results imply differential metabolism between clones treated with conditions inductive and noninductive for flowering. Higher concentrations of putative GA₉ conjugate and free GA₉ in the hot and dry treatment indicate a higher capacity of synthesizing, for flowering, the physiologically important GA₄ in the heat and drought-treated material. This synthesis does not, however, result in a buildup of the GA₄ pool, probably because of a high turnover rate of GA₄. The cool and wet-treated material had higher amounts of GA₁ and GA₃, indicating that the differentiation was preferentially directed toward vegetative growth.     

Sulfhydryl Compounds in Melanocytes of Yellow (Ay/a ), Nonagouti (a/a), and Agouti (A/A) Mice

CLEFFMANN (1953, 1963a,b) has reported that yellow but not black melanocytes of agouti (A/A) rabbits contained reducing sulfhydryl compounds. We have attempted to repeat CLEFFMANN's observations in mouse melanocytes of the lethal yellow (Ay/a), nonagouti (a/a) and agouti (A/A) genotypes. Our results contradict those of CLEFFMANN and reveal that yellow and black melanocytes, regardless of genotype, possess equivalent amounts of histochemically detectable sulfhydryl compounds. These results do not support the hypothesis that agouti-locus genes act by controlling the sulfhydryl metabolism of pigment cells.     

Purification and characterization of a poly(A)-binding protein from chickpea (Cicer arietinum) epicotyl

A poly(A)-binding protein (PABP) with mol wt 72,000 has been purified from chickpea (Cicer arietinum) epicotyls by ammonium sulfate fractionation, Cibacron blue F3-GA and poly(A) agarose chromatography. The binding properties and the specificity of binding show that the purified protein is an analogue of PABPs in other eukaryotes. This PABP is highly susceptible to proteolysis and upon degradation forms a polypeptide fragment of mol wt 21,000 which has an independent poly(A) binding activity.     

A reaction between captopril (a high blood pressure drug) and gold(I)-thiomalate

A reaction of gold(I) thiomalates [Au(tm)], "Myocrisin" (an antiarthritic drug), with captopril (a high blood pressure drug) was carried out in aqueous solution at pH 7.20 using 13C NMR spectroscopy. Captopril, which exists in the cis (c) and trans (t) isomer forms, binds strongly with gold(I), ejecting thiomalate (Htm) as free ligand into solution.