The structure of B-helical C-G-A-T-C-G-A-T-C-G and comparison with C-C-A-A-C-G-T-T-G-G. The effect of base pair reversals

The crystal structure of the DNA decamer C-G-A-T-C-G-A-T-C-G has been solved to a resolution of 1.5 A, with a final R-factor of 16.1% for 5,107 two-sigma reflections. Crystals are orthorhombic space group P2(1)2(1)2(1), with cell dimensions a = 38.93 A, b = 39.63 A, c = 33.30 A, and 10 base pairs/asymmetric unit. The final structure contains 404 DNA atoms, 142 water molecules treated as oxygen atoms, and two Mg(H2O)6(2+) complexes. Decamers stack atop one another to simulate continuous helical columns through the crystal, as with three previously solved monoclinic decamers, but the lateral contacts between columns are quite different in the orthorhombic and monoclinic cells. Narrow and wide regions of the minor groove exhibit a single spine or two ribbons of hydration, respectively, and the minor groove is widest when BII phosphate conformations are opposed diagonally across the groove. Phosphate conformation, in turn, appears to have a base sequence dependence. Twist, rise, cup, and roll are linked as has been observed in the three monoclinic decamers and can be characterized by high or low twist profiles. In all five known decamer crystal structures and eight representative dodecamers, a high twist profile is observed with G-C and G-A steps whereas all other R-R steps are low twist profiles (R = purine). A-T and A-C steps are intermediate in character whereas C-A and C-G exhibit behavior that is strongly influenced by the profiles of the preceding and following steps. When sufficient data are in hand, sequence/structure relationships for all helix parameters probably should be considered in a 4-base pair context. At this stage of limited information the problem is compounded because there are 136 unique 4-base steps x-A-B-y in a double helix as compared with only 10 2-base steps A-B.     

Climatic sensitivity of species’ vegetative and reproductive phenology in a Hawaiian montane wet forest

Understanding how tropical tree phenology (i.e., the timing and amount of seed and leaf production) responds to climate is vital for predicting how climate change may alter ecological functioning of tropical forests. We examined the effects of temperature, rainfall, and photosynthetically active radiation (PAR) on seed phenology of four dominant species and community-level leaf phenology in a montane wet forest on the island of Hawaiʻi using monthly data collected over ~ 6 years. We expected that species phenologies would be better explained by variation in temperature and PAR than rainfall because rainfall at this site is not limiting. The best-fit model for all four species included temperature, rainfall, and PAR. For three species, including two foundational species of Hawaiian forests (Acacia koa and Metrosideros polymorpha), seed production declined with increasing maximum temperatures and increased with rainfall. Relationships with PAR were the most variable across all four species. Community-level leaf litterfall decreased with minimum temperatures, increased with rainfall, and showed a peak at PAR of ~ 400 μmol/m²s⁻¹. There was considerable variation in monthly seed and leaf production not explained by climatic factors, and there was some evidence for a mediating effect of daylength. Thus, the impact of future climate change on this forest will depend on how climate change interacts with other factors such as daylength, biotic, and/or evolutionary constraints. Our results nonetheless provide insight into how climate change may affect different species in unique ways with potential consequences for shifts in species distributions and community composition.     

Structure of the precursor to an enzyme mediating COOH-terminal amidation in peptide biosynthesis

Many bioactive peptides terminate with an amino acid alpha-amide at their COOH terminus. The enzyme responsible for this essential posttranslational modification is known as peptidyl-glycine alpha-amidating monooxygenase or PAM. We identified cDNAs encoding the enzyme by using antibodies to screen a bovine intermediate pituitary lambda gt11 expression library. Antibodies to a beta-galactosidase/PAM fusion protein removed PAM activity from bovine pituitary homogenates. The 108,207 dalton protein predicted by the complete cDNA is approximately twice the size of purified PAM. An NH2-terminal signal sequence and short propeptide precede the NH2 terminus of purified PAM. The sequences of several PAM cyanogen bromide peptides were localized in the NH2-terminal half of the predicted protein. The cDNA encodes an additional 430 amino acid intragranular domain followed by a putative membrane spanning domain and a hydrophilic cytoplasmic domain. The forms of PAM purified from bovine neurointermediate pituitary may be generated by endoproteolytic cleavage at a subset of the 10 pairs of basic amino acids in the precursor. High levels of PAM mRNA were found in bovine pituitary and cerebral cortex. In corticotropic tumor cells, levels of PAM mRNA and pro-ACTH/endorphin mRNA were regulated in parallel by glucocorticoids and CRF.     

Characteristics of dynamic postural reactions in the locust hindleg

Summary The use of the locust (Schistocerca americana) hindleg in postural control was examined in animals that stood on a repeatedly swayed vertical substrate. Myograms were recorded from leg muscles and the angle of the femoro-tibial joint was monitored photographically. Two discrete strategies were observed,; in compensatory reactions the hindleg was held in place, while in flexion reactions, the leg was moved, most often to complete flexion of the femoro-tibial joint. Tightly coupled, rhythmic bursting occurred in the flexor and levator muscles of the leg during compensatory reactions. Bursting was initiated repeatedly when the substrate was being pulled away from the animal. Bursting was correlated with subsequent decreases in the rate of change of the femorotibial joint angle. Compensatory and flexion reactions occurred preferentially in different ranges of joint angles: most often, compensatory reactions occurred in the midrange, while flexion reactions were elicited in the extremes of joint angle. These differences may be due to the mechanical advantages of the tibial muscles and the leg may be moved to full flexion because of a locking mechanism of the flexor muscle tendon. These reactions are compared with known reflexes of hindleg proprioceptors and contrasted with similar responses of vertebrates.     

Binding of an antitumor drug to DNA, Netropsin and C-G-C-G-A-A-T-T-BrC-G-C-G

The antitumor antibiotic netropsin has been co-crystallized with a double-helical B-DNA dodecanucleotide of sequence: C-G-C-G-A-A-T-T-BrC-G-C-G, and the structure of the complex has been solved by X-ray diffraction at a resolution of 2.2 A. The structure has been refined independently by Jack-Levitt and Hendrickson-Konnert least-squares methods, leading to a final residual error of 0.257 by the Jack-Levitt approach (0.211 for two-sigma data) or 0.248 by the Hendrickson-Konnert approach, with no significant difference between refined structures. The netropsin molecule displaces the spine of hydration and fits snugly within the minor groove in the A-A-T-T center. It widens the groove slightly and bends the helix axis back by 8 degrees, but neither unwinds nor elongates the double helix. The drug molecule is held in place by amide NH hydrogen bonds that bridge adenine N-3 and thymine O-2 atoms, exactly as with the spine of hydration. The requirement of A X T base-pairs in the binding site arises because the N-2 amino group of guanine would demand impermissibly close contacts with netropsin. It is proposed that substitution of imidazole for pyrrole in netropsin should create a family of "lexitropsins" capable of reading G X C-containing base sequences.     

The Molecular through Ecological Genetics of Abnormal Abdomen in DROSOPHILA MERCATORUM. I. Basic Genetics

The abnormal abdomen syndrome (aa) in Drosophila mercatorum is characterized by the persistence of juvenilized cuticle on the adult abdomen. The aa phenotype is shown to depend on at least two X-linked genetic elements that are about one map unit apart near the centromeric end of the X chromosome. These two genetic elements are necessary for aa expression; one behaves as a dominant element and the other as a recessive. Overlaying these genetic studies upon molecular work reported elsewhere, it is argued that the dominant element is the presence of a 5 kb insertion in a majority of the X-linked repeats coding for the 28S ribosomal RNA. The recessive element appears to be a locus controlling differential replication of noninserted over inserted 28S genes during polytenization. The aa syndrome requires both the presence of the inserted repeats and the failure to preferentially amplify noninserted repeats. Given the necessary X-linked elements for aa, a variety of modifiers are revealed. First, aa expression in males is Y-linked, apparently corresponding to a deletion of the 18S/28S rDNA gene cluster normally found on the Y. Moreover, all major autosomes can modify the penetrance of aa.     

Helix geometry and hydration in an A-DNA tetramer: IC-C-G-G

The DNA oligomer of sequence IC-C-G-G has been synthesized, and its X-ray crystal structure solved at a resolution of 2.0 A, using anomalous scattering from iodines in phase analysis: 48 cycles of Jack-Levitt restrained least-squares refinement resulted in a residual error of 19.9% over all data, or 16.5% for two-sigma data. Two double-helical tetramers stack in the crystal to form a continuous octamer, except for the two missing phosphate connections across the center. The octamer has a mean helix rotation of 33.7 degrees (10.7 base-pairs per turn), rise of 2.87 A, mean inclination angle of base-pairs of 14 degrees, and mean base-pair propeller twist of +16.3 degrees. Local variations in both helix rotation and base plane roll angles, including those across the center of the octamer, are as predicted from base sequence by sum functions sigma 1 and sigma 2. The three known DNA octamers: IC-C-G-G/IC-C-G-G, G-G-T-A-T-A-C-C and G-G-C-C-G-G-C-C, make up a graded series in this order, with monotonically changing structural parameters. An exhaustive comparison of torsion angle correlations among the known A helices confirms some structural expectations and reveals some new features. 86 water molecules have been located per double-helical IC-C-G-G tetramer (the asymmetric unit), of which 451/2 per tetramer lie within a first hydrogen-bonded shell of hydration. No ordered water structure is observed comparable to the minor groove spine of hydration in B-DNA.     

Tetrahymena pyriformis as a protective antigen against Ichthyophthirius multifiliis infection: comparisons between isolates and ciliary preparations

Channel catfish, Ictalurus punctatus , were immunized with cilia from three isolates of Tetrahymena pyriformis and challenged with Ichthyophthirius multifiliis using a reproducible quantitative procedure. Two different methods of deciliation were used in antigen preparation. Results indicate that T. pyriformis cilia elicit an immune response in channel catfish against I. multifiliis , and that the protective ability of the cilia varies between T. pyriformis strains.     

Two-Phase Slug Flow Heat Exchanger for Microbial Thermal Inactivation Research

A continuous two-phase (air-liquid), slug flow, tubular heat exchanger was developed for microbial thermal inactivation research to give exposure times and temperatures in the range of high-temperature, short-time milk pasteurization as well as heat-treated sample volumes of at least 2 ml. The use of air to compartmentalize the liquid in the capillary tubing prevented the development of laminar flow, which enabled precise identification of the residence time of the fastest flowing particles in the heating, holding, and cooling sections of the instrument. Residence time distributions were quantitated by measuring the degree of spreading of radioactive tracers for water, whole milk, chocolate milk, cream, and ice-cream mix with holding temperatures from 50 to 72 C, holding times from 2 to 60 sec, and heating and cooling times of 1.7 sec each. For a holding time of 60 sec and a fastest particle velocity of 10.2 cm/sec, the velocity ratios of the fastest moving particle to the median particle were 1.05, 1.05, 1.10, and 1.13 for whole milk, chocolate milk, cream, and ice-cream mix, respectively. With shorter holding times, these velocity ratios were even closer to unity. These velocity ratios indicated that the instrument would be an effective tool for thermal inactivation research on microorganisms suspended in homogeneous fluids with a viscosity of 15 centipoises or less at the exposure temperature.