Proline-containing beta-turns. IV. Crystal and solution conformations of tert.-butyloxycarbonyl-L-prolyl-D-alanine and tert.-butyloxycarbonyl-L-prolyl-D-alanyl-L-alanine

The conformations of the dipeptide t-Boc-Pro-DAla-OH and the tripeptide t-Boc-Pro-DAla-Ala-OH have been determined in the crystalline state by X-ray diffraction and in solution by CD, n.m.r. and i.r. techniques. The unit cell of the dipeptide crystal contains two independent molecules connected by intermolecular hydrogen bonds. The urethane-proline peptide bond is in the cis orientation in both the molecular forms while the peptide bond between Pro and DAla is in the trans orientation. The single dipeptide molecule exhibits a "bent" structure which approximates to a partial beta-turn. The tripeptide adopts the 4----1 hydrogen-bonded type II beta-turn with all trans peptide bonds. In solution, the CD and i.r. data on the dipeptide indicate an ordered conformation with an intramolecular hydrogen bond. N.m.r. data indicate a significant proportion of the conformer with a trans orientation at the urethane-proline peptide bond. The temperature coefficient of the amide proton of this conformer in DMSO-d6 points to a 3----1 intramolecular hydrogen bond. Taken together, the data on the dipeptide in solution indicate the presence (in addition to the cis conformer) of a C7 conformation which is absent in the crystalline state. The spectral data on the tripeptide indicate the presence of the type II beta-turn in solution in addition to the nonhydrogen-bonded conformer with the cis peptide bond between the urethane and proline residues. The relevance of these data to studies on the substrate specificity of collagen prolylhydroxylase is pointed out.     

Chloroplast Genetics of Chlamydomonas. III. Closing the Circle

A novel mapping procedure is presented for organelle genes or any other genetic system exhibiting a measurable frequency of exchanges occurring at a constant rate over a measurable time interval. For a set of markers in a multiply-marked cross, the exchange rates measure relative map distances from a centromere-like attachment point.With this method, we present mapping data and a linear map of genes in the chlcroplast genome of Chlamydomonas. The data are plotted as log (percent remaining heterozygotes) against time and map distances are taken as proportional to slope.A statistical method which is an adaptation of jackknife methodology to a regression problem was developed to estimate slope values. A single line is fitted to pooled data for each marker from several crosses, and then lines are re-fit to a series of pooled data sets in each of which the observations from a single cross have been omitted. From these data sets a final summary slope is computed as well as a statement of its variability. The relative positions of new markers present in single crosses can then be estimated utilizing data from many crosses. The method does not distinguish between one-armed and two-armed linear or circular maps. However, evaluation of this map in conjunction with cosegregation frequency data (Sager and Ramanis 1976b) provides unambiguous evidence of the genetic circularity of the Chlamydomonas chloroplast genome.     

Receptor-mediated cell attachment and detachment kinetics. II. Experimental model studies with the radial-flow detachment assay.

Quantitative information regarding the kinetics of receptor-mediated cell adhesion to a ligand-coated surface are crucial for understanding the role of certain key parameters in many physiological and biotechnology-related processes. Here, we use the probabilistic attachment and detachment models developed in the preceding paper to interpret transient data from well-defined experiments. These data are obtained with a simple model cell system that consists of receptor-coated latex beads (prototype cells) and a Radial-Flow Detachment Assay (RFDA) using a ligand-coated glass disc. The receptors and ligands used in this work are complementary antibodies. The beads enable us to examine transient behavior with particles that possess fairly uniform properties that can be varied systematically, and the RFDA is designed for direct observation of adhesion to the ligand-coated glass surface over a range of shear stresses. Our experiments focus on the effects of surface shear stress, receptor density, and ligand density. These data provide a crucial test of the probabilistic framework. We show that these data can be explained with the probabilistic analyses, whereas they cannot be readily interpreted on the basis of a deterministic analysis. In addition, we examine transient data on cell adhesion reported from other assays, demonstrating the consistency of these data with the predictions of the probabilistic models.     

FlyBase--the Drosophila database. The FlyBase Consortium.

FlyBase is a database of genetic and molecular data concerning Drosophila. FlyBase is maintained as a relational database (in Sybase) and is available from the ftp.bio.indiana.edu Gopher server. The scope of FlyBase includes: genes, alleles, aberrations, pointers to sequence data, stock lists, Drosophila workers and bibliographic references.     

Multiple gene sequences delimit Botryosphaeria australis sp. nov. from B. lutea

Botryosphaeria lutea (anamorph Fusicoccum luteum) most easily is distinguished from other Botryosphaeria spp. by a yellow pigment that is formed in young cultures. This fungus has been reported from a number of cultivated hosts in New Zealand and Portugal. During a survey of Botryosphaeria fungi that occur on native Acacia species in Australia, a yellow pigment was observed in some cultures. These isolates were morphologically similar to B. lutea, but the pigment differed slightly from the one formed by authentic B. lutea isolates. Preliminary data also revealed small differences in ITS rDNA sequence data. The aim of this study was to determine whether these small differences were indicative of separate species or merely variations within B. lutea. Anamorph, teleomorph and culture morphology were compared between B. lutea and Acacia isolates from Australia. Sequence data of two other genome regions, namely the β-tubulin and EF1-α gene and intron regions, were combined with ITS rDNA sequence data to determine the phylogenetic relationship between these isolates. Isolates of B. lutea and those from Australian Acacia species were not significantly different in spore morphology. The yellow pigment, however, was much more distinct in cultures of B. lutea than in cultures from Acacia. There were only a few base pair variations in each of the analyzed gene regions, but these variations were fixed in the two groups in all regions. By combining these data it was clear that B. lutea and the isolates from Acacia were distinct species, albeit very closely related. We, therefore, propose the new epithet B. australis for the fungus from Australia. Botryosphaeria australis also was isolated in this study from exotic Sequoiadendron trees in Australia. Re-analyses of GenBank data in this study showed that B. australis also occurs on other native Australian hosts, namely a Banksia sp. and a Eucalyptus sp., as well as a native Protea sp. in South Africa and on Pistachio in Italy. These records from GenBank have been identified previously as B. lutea. The common occurrence of B. australis on a variety of native hosts across Australia suggests that this fungus is native to this area.     

Interpretive reporting of laboratory data. Gastric analysis.

The reporting of gastric analysis data requires clearly formatted and documented analytic results, accurately calculated secretory rates, appropriate normal values based on age, sex and weight of the patient, and interpretive review of the data based on well-documented personal experience or comprehensive studies reported in the medical literature. We report here a technique using inexpensive and flexible data processing equipment suitable to most community hospital laboratories.     

Gating current harmonics. I. Sodium channel activation gating in dynamic steady states.

Internally perfused and pronase-treated giant axons were prepared for gating current measurements. Gating current records were obtained under large-amplitude sinusoidal voltage clamp after allowing for settling times into dynamic steady states. The current records were analyzed as functions of the mean membrane potential of the test sinusoid for which the amplitude and frequency were held constant. The nonlinear analysis consisted of determining the harmonic content (amplitudes and phases) of the distorted periodic current records. The most pronounced feature found in the analysis is a dominant second harmonic centered at Emean = +10 mV. A number of other characteristic harmonic behaviors were also observed. The harmonics tend to die away for very small (less than -60 mV) and very large (greater than +72 mV) values of Emean. The harmonic behavior seen in the axonal data is basically different from that seen in gating current simulations generated by the sodium-activation kinetics of standard models, including the Hodgkin-Huxley model. Some of the differences can be reconciled without requiring fundamental changes in the model kinetic schemes. However, the dominant harmonic feature seen in the axonal data cannot be reconciled with the model kinetics without a fundamental change in the models. The axonal data suggest two moving molecular components with independent degrees of freedom whose properties are outlined on the basis of the data presented herein.     

A new analysis for membrane noise. The integral spectrum.

A new method of random data analysis has been developed with special implications for membrane noise. The integral spectrometer uses overlapping broad-band filters of simple design, whose bandwidth increases linearly with center frequency. A random two-state process, which has a Lorentzian-shaped spectral density, results in an integral spectrum whose maximum value occurs at the mean frequency of the events, and which is symmetric about that frequency on a semilog plot. 1/f noise is flat and does not distort the symmetry on the frequency axis. The integral spectrum exchanges resolution on the frequency axis for accuracy in the amplitude. The expected statistical error in amplitude has been calculated for three types of membrane noise assuming finite data. The integral spectrum compares favorably with conventional spectral densities and may be a reasonable alternative for random data analysis.     

P.T.C. thresholds, blood factors, colour vision and fingerprints of Jivaro Indians in Eastern Ecuador

Among the Jivaro Indians of Eastern Ecuador, very low non-taster frequencies were found, a situation in accord with most of the other, through few sets of P. T. C. data available for South American Indians. The Jivaro have maximal frequencies (1.000) of the alleles I° and D, typical of most South American Indian groups. The colour blindness frequency of males (7.1%) is seemingly higher than North American Indian figures and is similar to European frequencies. Jivaro dermatoglyphic data are in broad general agreement with data available for Amerindian groups generally, and for many Asian mongoloids, with high pattern intensity indices for males and females, mainly the result of a very high whorl incidence. The digital distribution of the pattern types for the greater part accords with the generalisations of Cummins and Midlo ('61). There are marked sex differences in the data, females having a lower mean pattern intensity index, a higher value for Dankmeijer's Index and a lower value for Furuhata's Index. Unfortunately there are few other sets of South American Indian data available for comparative purposes.     

Waiting list statistics. III. Comparison of two measures of waiting times.

The length of time that patients spend on waiting lists is a topic of current concern. Calculating the proportion of patients who have been on a waiting list for a long time by taking a census of patients on the list at a single point in time will tend to yield a higher estimate than that obtained by calculating waiting times of patients admitted to hospital during a period of time. To illustrate this point the waiting times of patients in the Oxford region as measured by SBH 203 returns ("census" data) were compared with those as measured by the Hospital Activity Analysis ("event" data). As expected, the SBH 203 census returns showed a higher proportion of patients who had waited over a year compared with the "event" measure of all admissions. This difference, which is analagous to the difference between prevalence and incidence in epidemiology, should be considered when using data from these sources to calculate waiting times.     

On gaps.

Gaps result from the alignment of sequences of unequal length during primary homology assessment. Viewed as character states originating from particular biological events (mutation), gaps contain historical information suitable for phylogenetic analysis. The effect of gaps as a source of phylogenetic data is explored via sensitivity analysis and character congruence among different data partitions. Example data sets are provided to show that gaps contain important phylogenetic information not recovered by those methods that omit gaps in their calculations. However, gap cost schemes are arbitrary (although they must be explicit) and thus data exploration is a necessity of molecular analyses, while character congruence is necessary as an external criterion for hypothesis decision.     

Biophysical studies by ultracentrifugation.

Advances in data analysis are broadening the applicability of ultracentrifugation to the characterization of macromolecular behavior in complex solution. The direct fitting of sedimentation velocity data to the Lamm equation is emerging as a very powerful means to characterize size distributions, improve the precision of data analysis and increase experimental throughput. With improvements in data acquisition and analysis, ultracentrifugation is poised to make significant contributions to our understanding of how macromolecules behave in vivo.     

Time-resolved methods in biophysics. 6. Time-resolved Laue crystallography as a tool to investigate photo-activated protein dynamics.

When polychromatic X-rays are shined onto crystalline material, they generate a Laue diffraction pattern. At third generation synchrotron radiation sources, a single X-ray pulse of approximately 100 ps duration is enough to produce interpretable Laue data from biomolecular crystals. Thus, by initiating biological turnover in a crystalline protein, structural changes along the reaction pathway may be filmed by ultra-fast Laue diffraction. Using laser-light as a trigger, transient species in photosensitive macromolecules can be captured at near atomic resolution with sub-nanosecond time-resolution. Such pump-probe Laue experiments have now reached an outstanding level of sophistication and have found a domain of excellence in the investigation of light-sensitive proteins undergoing cyclic photo-reactions and producing stiff crystals. The main theoretical concepts of Laue diffraction and the challenges associated with time-resolved experiments on biological crystals are recalled. The recent advances in the design of experiments are presented in terms of instrumental choices, data collection strategy and data processing, and some of the inherent difficulties of the method are highlighted. The discussion is based on the example of myoglobin, a protein that has traversed the whole history of pump-probe Laue diffraction, and for which a massive amount of data have provided considerable insight into the understanding of protein dynamics.     

Design and analysis of experiments on mutagenicity. II. Assays involving microorganisms.

The design and statistical analysis of mutagenicity experiments involving microorganisms and a single dose of mutagen are discussed. Test statistics are derived for use in determining the mutagenicity of a chemical when survival data are available and also when such data are not available. One's likelihood (power) of correctly concluding a chemical is mutagenic is examined, and minimum total sample sizes required for 95% power are presented. It is found that one generally has greater power when survival data are available. Required precision is estimating survival is discussed in reference to type-1 and type-2 errors. The proper use of the formulae and figures presented is illustrated by examples.     

FlyBase: the Drosophila database. The Flybase Consortium.

FlyBase is a database of genetic and molecular data concerning Drosophila. FlyBase is maintained as a relational database (in Sybase). The scope of FlyBase includes: genes, alleles (and phenotypes), aberrations, pointers to sequence data, clones, stock lists, Drosophila workers and bibliographic references. FlyBase is also available on CD-ROM for Macintosh systems (Encyclopaedia of Drosophila).     

Introducing W.A.T.E.R.S.: a Workflow for the Alignment,Taxonomy, and Ecology of Ribosomal Sequences

Background  

For more than two decades microbiologists have used a highly conserved microbial gene as a phylogenetic marker for bacteria and archaea. The small-subunit ribosomal RNA gene, also known as 16 S rRNA, is encoded by ribosomal DNA, 16 S rDNA, and has provided a powerful comparative tool to microbial ecologists. Over time, the microbial ecology field has matured from small-scale studies in a select number of environments to massive collections of sequence data that are paired with dozens of corresponding collection variables. As the complexity of data and tool sets have grown, the need for flexible automation and maintenance of the core processes of 16 S rDNA sequence analysis has increased correspondingly.     

REPRODUCTIVE SEASONALITY OF WESTERN ATLANTIC BOTTLENOSE DOLPHINS OFF NORTH CAROLINA, U.S.A.

We describe reproductive seasonality of bottlenose dolphins in North Carolina (NC), U.S.A., using strandings data from the entire coast of NC and sighting data from Beaufort, NC and by estimating dates of birth of known females. We found a strong peak of neonate strandings in the spring (April-May), and low levels of neonate strandings in the fall and winter. The distribution of neonate strandings was significantly different from a uniform distribution ( P < 0.001, K = 3.8). We found a unimodal distribution of 282 sightings of neonates with a diffuse peak in the summer. The temporal distribution of sightings of neonates departed significantly from a uniform distribution (P < 0.001, K = 5.1). Estimated birth dates of neonates from known females occurred in May ( n = 6) and June ( n = 4), with a single fall birth. These methods shed light on bottlenose reproductive patterns and underscore the value of using information from multiple types of data. Clarification of bottlenose dolphin reproductive patterns, such as the seasonality of birth, may enhance our understanding of the population structure of this species in the mid-Atlantic region.