Relations between synthesis of deoxyribonucleotides and DNA replication in 3T6 fibroblasts

In exponentially growing 3T6 cells, the synthesis of deoxythymidine triphosphate (dTTP) is balanced by its utilization for DNA replication, with a turnover of the dTTP pool of around 5 min. We now investigate the effects of two inhibitors of DNA synthesis (aphidicolin and hydroxyurea) on the synthesis and degradation of pyrimidine deoxynucleoside triphosphates (dNTPs). Complete inhibition of DNA replication with aphidicolin did not decrease the turnover of pyrimidine dNTP pools labeled from the corresponding [3H]deoxynucleosides, only partially inhibited the in situ activity of thymidylate synthetase and resulted in excretion into the medium of thymidine derived from breakdown of dTTP synthesized de novo. These data demonstrate continued synthesis of dTTP in the absence of DNA replication. In contrast, hydroxyurea decreased the turnover of pyrimidine dNTP pools 5-50-fold. Hydroxyurea is an inhibitor of ribonucleotide reductase and stops DNA synthesis by depleting cells of purine dNTPs but not pyrimidine dNTPs. Our results suggest that degradation of dNTPs is turned off by an unknown mechanism when de novo synthesis is blocked.     

Sequence heterogeneity and anomalous electrophoretic mobility of kinetoplast minicircle DNA from Leishmania tarentolae

Several unit-length minicircles from the kinetoplast DNA of Leishmania tarentolae were cloned into pBR322 and into M13 phage vectors. The complete nucleotide sequences of three different partially homologous minicircles were obtained. The molecules contained a region of approx. 80% sequence homology extending for 160–270 bp and a region unique to each minicircle. A 14-mer was found to be conserved in all kinetoplast minicircle sequences reported to date. The frequency distributions of various minicircle sequence classes in L. tarentolae were obtained by quantitative gel electrophoresis and by examination of the “T ladder” patterns of minicircles randomly cloned into M13 at several sites. By these methods we could assign approx. 50% of the total minicircle DNA into a minimum of five sequence classes. A sequence-dependent polyacrylamide gel migration abnormality was observed with several minicircle fragments both cloned and uncloned. The abnormality was dependent on the presence of a portion of the conserved region of the minicircle.     

Behavioral and Emotional Dynamics of Two People Struggling to Reach Consensus about a Topic on Which They Disagree

We studied the behavioral and emotional dynamics displayed by two people trying to resolve a conflict. 59 groups of two people were asked to talk for 20 minutes to try to reach a consensus about a topic on which they disagreed. The topics were abortion, affirmative action, death penalty, and euthanasia. Behavior data were determined from audio recordings where each second of the conversation was assessed as proself, neutral, or prosocial. We determined the probability density function of the durations of time spent in each behavioral state. These durations were well fit by a stretched exponential distribution, with an exponent, , of approximately 0.3. This indicates that the switching between behavioral states is not a random Markov process, but one where the probability to switch behavioral states decreases with the time already spent in that behavioral state. The degree of this “memory” was stronger in those groups who did not reach a consensus and where the conflict grew more destructive than in those that did. Emotion data were measured by having each person listen to the audio recording and moving a computer mouse to recall their negative or positive emotional valence at each moment in the conversation. We used the Hurst rescaled range analysis and power spectrum to determine the correlations in the fluctuations of the emotional valence. The emotional valence was well described by a random walk whose increments were uncorrelated. Thus, the behavior data demonstrated a “memory” of the duration already spent in a behavioral state while the emotion data fluctuated as a random walk whose steps did not have a “memory” of previous steps. This work demonstrates that statistical analysis, more commonly used to analyze physical phenomena, can also shed interesting light on the dynamics of processes in social psychology and conflict management.     

Affinity chromatography on anti-B1 monoclonal gels for purification and characterization of protein B1 from Escherichia coli ribonucleotide reductase

Affinity gels were prepared from four monoclonal antibodies against the B1 protein of ribonucleotide reductase of Escherichia coli. The gels were used to purify protein B1 and also to study some of its properties. Gels from the nonneutralizing monoclonal anti-B1-k bound as much as 2 mg of B1/mL and were employed to prepare essentially pure B1 protein in a single step from extracts of wild-type E. coli and strains overproducing the subunit. However, B1 prepared from wild-type extracts had a lowered specific activity, suggesting some denaturation during elution of the protein from the column. Addition of the allosteric effector dATP during affinity chromatography changed the chromatographic pattern. Some protein B2, the second subunit of the reductase, remained in all cases bound to the gels together with B1. The gel prepared from anti-B1-c retained two additional proteins. In other experiments involving binding of proteolytic fragments of B1 to various antibodies, we also found a striking effect of dATP, suggesting that dATP made protein B1 less accessible to proteolysis. In these experiments fragments around 15K still had the ability to bind monoclonals, making possible more detailed investigations of the structural contacts between B1 and the monoclonals.     

cDNA and protein sequence of a major pea seed albumin (PA 2 : Mr≈26 000)

Summary Pea albumin 2 (PA2:M_r26000) is a major component of the albumin fraction derived from aqueous salt extracts of pea seed. Sodium dodecylsulfate-polyacrylamide gel electrophoresis and chromatography on DEAE-Sephacel resolve PA2 into two closely related components (PA2a and PA2b). A cDNA clone coding for one of these components has been sequenced and the deduced amino acid sequence compared with partial, chemically-determined sequences for cyanogen bromide peptides from both PA2 components. Complete amino acid sequences were obtained for the C-terminal peptides. The PA2 molecule of 230 amino acids contains four imperfect repeat sequences each of approximately 57 amino acids in length.The combined sequence data, together with a comparison of PA2-related polypeptides produced in vitro and in vivo, indicate that PA2 is synthesized without a signal sequence and does not undergo significant post-translational modification. Although both forms of PA2 contain Asn-X-Thr consensus sequences, neither form is glycosylated. Accumulation of PA2 contributes approximately 11% of the sulfur-amino acids in pea seeds (cysteine plus methionine equals 2.6 residues percent). Suppression of levels of PA2 polypeptides and their mRNAs in developing seeds of sulfur-deficient plants is less marked than that for legumin, in spite of the lower content of sulfur-amino acids in legumin.     

NAD(P)H:flavin oxidoreductase of Escherichia coli. A ferric iron reductase participating in the generation of the free radical of ribonucleotide reductase

The active form of one subunit of Escherichia coli ribonucleotide reductase (protein B2) contains an organic free radical localized to tyrosine 122 of its polypeptide chain. When this radical is scavenged, e.g. by treatment with hydroxyurea, the enzyme is inactivated (protein B2/HU). E. coli contains an enzyme system consisting of at least three proteins that in the presence of NADPH, FMN, dithiothreitol, and oxygen introduce the tyrosyl radical into B2/HU (Eliasson, R., J?rnvall, H., and Reichard, P. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 2373-2377). One of the three proteins was identified as superoxide dismutase. We now identify a second protein, previously provisionally named Fraction c, as an NAD(P)H:flavin oxidoreductase (flavin reductase). After 4,000-fold purification the protein moved as a single band on sodium dodecyl sulfate gel electrophoresis with a molecular weight of 28,000-29,000. The enzyme contained no flavin but reduced riboflavin, FMN, and FAD by NADH, or riboflavin and FMN by NADPH. It is a powerful ferric iron reductase. We propose that its complementing activity during radical generation involves participation in the reduction of the ferric iron center of protein B2/HU. Radical formation is then linked to the reoxidation of iron by oxygen. The flavin reductase may also participate in other aspects of iron metabolism of E. coli.     

Isolation of Different Agrobacterium Biovars from a Natural Oak Savanna and Tallgrass Prairie

Populations of agrobacteria in excess of 10⁵ CFU/g were recovered from 12 soil and root samples obtained from the Allison Savanna, Minn., a natural oak savanna and tallgrass prairie which has never been disturbed agriculturally. Of 126 strains picked randomly from selective media, 54 were identified as Agrobacterium spp. Biovar 2 strains predominated (35 of 54), but these strains were distributed into three phenotypically distinct subgroups. Of the remaining Agrobacterium strains, four were biovar 1-2, one was biovar 1, and none were biovar 3. The last 14 Agrobacterium strains formed a homogeneous group which differed biochemically from the hitherto reported biovars. Opine utilization (coded for by genes on the tumor-inducing plasmid in pathogenic Agrobacterium spp.) by these agrobacteria was limited to two biovar 2 strains. In contrast, 10 nonfluorescent gram-negative strains utilized either nopaline or octopine as the sole carbon and nitrogen source. There may be a need to reexamine the source and role of opines in the terrestrial environment because (i) all of these opine utilizers were isolated from an environment free of crown gall, the only known terrestrial source of opines, and (ii) 83% of the opine utilizers were not Agrobacterium spp.