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.     

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.     

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.     

Complementary Methodologies To Identify Specific Agrobacterium Strains

Serological techniques and restriction enzyme cleavage patterns of total DNA were used to differentiate strains of Agrobacterium spp. Forty-five wild-type and plasmid-cured Agrobacterium strains were tested by immunodiffusion and immunofluorescence against polyclonal antisera to a crude ribosome preparation from Agrobacterium strains K84, U11, B6, A323, NT1, and C58. In immunodiffusion gels, these antisera reacted only with water-phenol extracts of the homologous strain, producing a single, strain-specific precipitin line. In contrast, when the same antisera were used in immunofluorescence staining, cross-reactions occurred with a limited number of heterologous Agrobacterium strains. However, the cross-reacting heterologous cells fluoresced generally less brightly than the homologous cells. When the EcoRI-digested DNA profiles from the same Agrobacterium strains were compared, 34 distinct cleavage patterns were observed. The DNA profiles were the same for all strains sharing a common chromosomal background and correlated with the strain-specific serological reaction. The presence or absence of plasmid DNA did not alter the strain-specific serological reaction or the DNA cleavage patterns. Both the serological reaction and the restriction enzyme digestion of total DNA were complementary to each other. These methods were used successfully to identify A. radiobacter K84 strains which were recovered 6 months after being inoculated to young trees in the field.     

Hydrolysis of dilute acid pretreated mixed hardwood and purified microcrystalline cellulose by cell-free broth from Clostridium thermocellum

The cellulase activity in cell-free broths from the thermophilic, ethanol-producing anaerobic bacterium Clostridium thermocellum is examined on both dilute-acid-pretreated mixed hardwood (90% maple, 10% birch) and Avicel. Experiments were conducted in vitro in order to distinguish properties of the cellulase from properties of the organism and to evaluate the effectiveness of C. thermocellum cellulase in the hydrolysis of a naturally occurring, lignin-containing substrate. The results obtained establish that essentially quantitative hydrolysis of cellulose from pretreated mixed hardwood is possible using this enzyme system. Pretreatment with 1% H(2)SO(4) and a 9-s residence time at 220, 210, 200, and 180 degrees C allowed yields after enzymatic hydrolysis (percentage of glucan solubilized/ glucan potentially solubilized) of 97.8, 86.1, 82.0, and 34.6%, respectively. Enzymatic hydrolysis of mixed hardwood with no pretreatment resulted in a yield of 10.1%. Hydrolysis yields of >95% were obtained from approximately 0.6 g/L mixed hardwood pretreated at 220 degrees C in 7 h at broth strengths of 60 and 80% (v/v) and in approximately 48 h with 33% broth. Hydrolysis of pretreated mixed hardwood is compared to hydrolysis of Avicel, a pure microcrystalline cellulose studied previously. The initial rate of Avicel hydrolysis saturates with respect to enzyme, whereas the initial rate of hydrolysis of pretreated wood is proportional to the amount of enzyme present. Initial hydrolysis rates for pretreated wood and Avicel at 0.6 g/L are greater for wood at low broth dilutions (1.25: 1 to 5 :1) by up to 2.7-fold and greater for Avicel at high broth dilutions (5 : 1 to 50 : 1) by up to 4.3-fold. Maximum rates of hydrolysis are achieved at <2 g substrate/L for both pretreated wood and Avicel. The substrate concentration at one-half the maximum observed rate for C. thermocellum broths is smaller for pretreated mixed hardwood than for Avicel and decreases with increasing broth dilution for both substrates. An initial activity per volume broth of approximately 11 mumol soluble glucose equivalent produced/L broth/min is observed for mixed hardwood pretreated at 220 degrees C and for Avicel at high broth dilutions; the initial activity per volume broth for Avicel is lower at low broth dilutions. The results indicate that pretreated wood is hydrolyzed at rates comparable to Avicel under many conditions and at rates significantly faster than Avicel under several conditions.     

DNA replication in maize leaf protoplasts

Maize leaf protoplasts were investigated for their metabolic competence and capacity to synthesize DNA. When protoplasts were incubated at elevated temperatures, they exhibited a heat shock response with specific proteins being preferentially synthesized. This indicated that the protoplasts were fully metabolically functional and capable of responding to environmental stimuli. Significant DNA synthesis was observed in these protoplasts after incorporation of ³H-thymidine into chromatin by trichloroacetic acid precipitation and by incorporation of 5-bromo-2-deoxyuridine (BrdU), an analog of thymidine, detected by immunofluorescence. The immunocytochemical method revealed that about 50% of nuclei in the maize leaf protoplasts were labelled after 3 days of culture and that most of these nuclei were labelled as intensely as normal mitotic cells. Aphidicolin, an inhibitor of DNA polymerase-, decreased the percentage of labelled nuclei, demonstrating that the labelling was substantially due to replicative DNA synthesis. However, chromosome condensation was not observed. It is proposed that these protoplasts are capable of DNA synthesis, but incapable of nuclear division. Effects of media additives on the number of nuclei entering S phase in these protoplasts were also assessed by the immunocytochemical method. Inclusion of 80mM Ca²⁺ in the enzyme solution increased protoplast yield and also appeared beneficial to DNA synthesis. The antioxidant, n-propyl gallate, which was used to stabilize the protoplasts, delayed the onset of DNA synthesis. Arginine and spermidine produced a slight increase in DNA synthesis.Abbreviations BrdU 5-bromo-2-deoxyuridine - DMSO dimethyl sulfoxide - n-PG n-propyl gallate - PBS phosphate-buffered saline Dedicated to Dr. Friedrich Constabel on the occasion of his 60th birthday