End Loops Research Articles

Studies of DNA dumbbells. IV. Preparation and melting of a DNA dumbbell with the 16 base‐pair sequence 5′G‐T‐A‐T‐C‐C‐C‐T‐C‐T‐G‐G‐A‐T‐A‐C3′ linked on the ends by dodecyl chains

The preparation and melting of a 16 base-pair duplex DNA linked on both ends by C12H24 (dodecyl) chains is described. Absorbance vs temperature curves (optical melting curves) were measured for the dodecyl-linked molecule and the same duplex molecule linked on the ends instead by T4 loops. Optical melting curves of both molecules were measured in 25, 55, and 85 mM Na+ and revealed, regardless of [Na+], the duplex linked by dodecyl loops is more stable by at least 6 degrees C than the same duplex linked by T4 loops. Experimental curves in each salt environment were analyzed in terms of the two-state and multistate theoretical models. In the two-state, or van't Hoff analysis, the melting transition is assumed to occur in an all-or-none manner. Thus, the only possible states accessible to the molecule throughout the melting transition are the completely intact duplex and the completely melted duplex or minicircle. In the multistate analysis no assumptions regarding the melting transition are required and the statistical occurrence of every possible partially melted state of the duplex is explicitly considered. Results of the analysis revealed the melting transitions of both the dodecyl-linked molecule and the dumbbell with T4 end loops are essentially two state in 25 and 55 mM Na+. In contrast, significant deviations from two-state behavior were observed in 85 mM Na+. From our previously published melting data of DNA dumbbells with Tn end loops where n = 2, 3, 4, 6, 8, 10, 14 [T. M. Paner, M. Amaratunga, and A. S. Benight, (1992) Biopolymers, Vol. 32, pp. 881-892] and the dumbbell with T4 end loops of this study, a plot of d(Tm)/d ln [Na+] was constructed. Extrapolation of this data to n = 1 intersects with the value of d (Tm)/d ln [Na+] obtained for the alkyl-linked dumbbell, suggesting the salt-dependent stability of the alkyl-linked molecule behaves as though the duplex of this molecule were linked by end loops comprised of a single T residue.

Studies of DNA dumbbells. V. A DNA triplex formed between a 28 base-pair DNA dumbbell substrate and a 16 base linear single strand.

CD spectra and melting curves were collected for a 28 base-pair DNA fragment in the form of a DNA dumbbell (linked on both ends by T4 single-strand loops) and the same DNA sequence in the linear form (without end loops). The central 16 base pairs (bp) of the 28-bp duplex region is the poly(pu) sequence: 5'-AGGAAGGAGGAAAGAG-3'. Mixtures of the dumbbell and linear DNAs with the 16-base single-strand sequence 5'-TCCTTCCTCCTTTCTC-3' were also prepared and studied. At 22 degrees C, CD measurements of the mixtures in 950 mM NaCl, 10 mM sodium acetate, 1 mM EDTA, pH 5.5, at a duplex concentration of 1.8 microM, provided evidence for triplex formation. Spectroscopic features of the triplexes formed with either a dumbbell or linear substrate were quite similar. Melting curves of the duplex molecules alone and in mixtures with the third strand were collected as a function of duplex concentration from 0.16 to 2.15 microM. Melting curves of the dumbbell alone and mixtures with the third strand were entirely independent of DNA concentration. In contrast, melting curves of the linear duplex alone or mixed with the third strand were concentration dependent. At identical duplex concentrations, the dumbbell alone melts approximately 20 degrees C higher than the linear duplex. The curve of the linear duplex displayed a significant pretransition probably due to end fraying. On melting curves of mixtures of the dumbbell or linear duplex with the third strand, a low temperature transition with much lower relative hyperchromicity change (approximately 5%) was observed. This transition was attributed to the melting of a new molecular species, e.g., the triplex formed between the duplex and single-strand DNA molecules. In the case of the dumbbell/single-strand mixture, these melting transitions of the triplex and the dumbbell were entirely resolvable. In contrast, the melting transitions of the linear duplex and the triplex overlapped, thereby preventing their clear distinction. To analyze the data, a three-state equilibrium model is presented. The analysis utilizes differences in relative absorbance vs temperature curves of dumbbells (or linear molecules) alone and in mixtures with the third strand.(ABSTRACT TRUNCATED AT 400 WORDS)

Structural features of a three-stranded DNA junction containing a C-C junctional bulge.

We have examined the stability of junctional base pairs in a three-way DNA junction with two unpaired cytidine residues at the branch point using two-dimensional nuclear Overhauser effect spectroscopy in H2O solution. Our data directly support the presence of two of the three junctional Watson-Crick base pairs, with indirect support for the third as well. These results complement the data presented in the preceding paper, where we examined the nonexchangeable proton resonance assignments of three-way DNA junctions from NOESY data in D2O solution. We have incorporated the NOE data from both sets of experiments, using this information as input for a combined distance geometry (DG) and simulated annealing (SA) protocol designed to derive three-dimensional structures of the junction molecule consistent with the NMR data. Although the data does not allow us to derive a unique solution for the structure of the molecule, certain conformational features are invariably present in our models. We demonstrate the existence of a preferred, pair-wise stacking arrangement between two of the three helices in the junction. Furthermore, the remaining duplex stem is situated so that it always forms an acute angle with just one of the arms from the quasi-continuous helix. The unpaired residues provide an extended backbone segment linking two of the helices together. The first unpaired base on the 5' end loops out from the interior of the molecule to reside along the minor groove of one helix. The second is located within the interior of the molecule, stacking below one of the junctional base pairs. Our findings suggest that junctional base pair stacking is an important determinant in the conformation of multistranded nucleic acid junctions. In three-way junctions, the presence of unpaired bases at the branch point provides a relief from covalent constraints that would otherwise prevent the simultaneous realization of both base pairing and base pair stacking within the branch point of the molecule.

Morphological observations on the unique paired capillaries of the opossum retina.

Light-microscopic and ultrastructural analysis of the ocular tissues of the North American opossum (Didelphis virginiana) revealed that the arterial and venous segments of retinal vessels, including capillaries of the smallest calibre, occur in pairs. They do not form anastomotic networks, the common pattern in mammals with vascularised retinae, but instead the two segments of the pair join to form hairpin end loops. The paired vessels, with the arteriolar limb usually on the vitread aspect, penetrate the retina and branch to form three distinct layers of capillaries. The most superficial lies in the nerve fiber layer, the middle is situated in the inner nuclear layer and the deepest extends to the external limiting membrane, which is considerably deeper than in normal mammalian holangiotic retinae. The paired capillaries display classical morphological features of central nervous system capillaries, i.e., they are lined by continuous endothelial cells united by tight junctions. The lining endothelium is supported by a distinct basal lamina that splits to envelop pericytes. The latter, although abundant, are invariably interposed between the two vessels that form each vascular unit. Phylogenetic and functional aspects of this unique form of retinal vascularisation are discussed.

Studies of DNA dumbbells. III. Theoretical analysis of optical melting curves of dumbbells with a 16 base-pair duplex stem and Tn end loops (n = 2, 3, 4, 6, 8, 10, 14).

Optical melting curves of seven DNA dumbbells with the 16 base-pair duplex sequence 5'G-C-A-T-A-G-A-T-G-A-G-A-A-T-G-C3' linked on both ends by Tn (n = 2, 3, 4, 6, 8, 10, and 14) loops measured in 30, 70, and 120 mM Na+ are analyzed in terms of the numerically exact statistical thermodynamic model of DNA melting. The construction and characterization of these molecules were described in the previous paper (Amaratunga et al., 1992). As was recently reported for hairpins (T. M. Paner, M. Amaratunga, M. J. Doktycz, and A. S. Benight, 1990, Biopolymers, Vol. 29, pp. 1715-1734) theoretically calculated melting curves were fitted to experimental curves by simultaneously adjusting the parameters representing loop and circle formation to optimize the fits. The systematically determined empirical parameters provide evaluations of the free energies of hairpin loop formation delta Gloop (n) and single-strand circles delta Gcircle (N), as a function of end loop size, n = 2-14, and circle size, N = 32 + 2n. The dependence of these quantities on solvent ionic strength over the range from 30 to 120 mM Na+ was evaluated. An approximately analytical expression for the partition function Q(T) of the dumbbells was formulated that allowed a means for determining the transition enthalpy delta H degrees and entropy delta S degrees for every dumbbell, revealing the dependence of these quantities on loop size. In this multistate approach a manifold of partially melted intermediate microstates are considered and therefore no assumptions regarding the nature of the melting transitions (that they are two-state) are required. The transition thermodynamic parameters were also determined from a van't Hoff analysis of the melting curves. Comparisons between the results of the multistate analysis and the two-state van't Hoff analysis revealed significant differences for the dumbbells with larger end loops, indicating that the melting transitions of the larger looped dumbbells deviate considerably from two-state behavior. Results are then compared with published melting studies of much larger DNA dumbbells (D. B. Naritsin and Y. L. Lyubchenko, 1990, Journal of Biomolecular Structure and Dynamics, Vol. 8, pp. 1-13), of small hairpins (Paner et al., 1990; M. J. Doktycz, T. M. Paner, M. Amaratunga and A. S. Benight, 1990, Biopolymers, Vol. 30, pp. 829-845) and another dumbbell (A. S. Benight, J. M. Schurr, P. F. Flynn, B. R. Reid, and D. E. Wemmer, 1988) Journal of Molecular Biology, Vol. 200, pp. 377-399).(ABSTRACT TRUNCATED AT 400 WORDS)

ヒト披裂筋の神経終末 銀染色法による観察

Light microscopic study of nerve endings in the human interarytenoid muscle was carried out by using the Baker's silver impregnated materials.In addition to the ordinary motor nerve endings, there were three types of nerve endings surrounding the muscle fibers in an annular or helical manner.The first type of the ending was composed of a thick myelinated fiber which tightly encircled and wrapped the muscle fibers. Branches from the myelinated fibers sporadically formed end buttons and loops. This type was determind as the spiral nerve ending.The second type of the ending had several muscle fibers possessing both the spiral nerve ending and motor nerve ending. This type of the ending was considered to be the muscle spindle.The third type of the ending was composed of both the spiral ending and motor nerve ending, which were thought to be either the nerve ending of the mixed type or the motor ending of the diffuse type.These were the additional findings to our first report that revealed the presence of muscle spindles in the intrinsic laryngeal muscles. A similar system has been found only in the outer ocular muscles.

Ultrastructural changes of the peripheral nerve induced by vibration: an experimental study.

To investigate the effects of vibration on the peripheral nerves, rabbits were exposed to vibration of 60 cycles/s frequency with 0.35 mm amplitude (acceleration: 51 m/s2) for two hours daily. After 150, 250, 450, and 600 hours vibration, thin sections of the saphenous and median nerves were examined under the electron microscope. Vibration was found to induce the following changes: (1) disruption of the myelin sheath and constriction of the axon, (2) accumulation of vacuoles in the nodal gap and paranodal region, (3) disorganisation of the paranodal end loops and detachment of the paranodal end loops from the axolemma, (4) dilatation of the Schmidt-Lanterman incisures (SLI) and increased density of SLI, and (5) disappearance of neurotubules and neurofilaments in axons. The diameters of myelin sheaths disrupted by vibration varied from 2 to 12 microns. The extent of the myelin disruption is proportional to the vibration dose.

Preparation and melting of single strand circular DNA loops.

A method for preparation of single strand DNA circles of almost arbitrary sequence is described. By ligating two sticky ended hairpins together a linear duplex is formed, closed at both ends by single stranded loops. The melting characteristics of such loops are investigated using optical absorbance and NMR. It is shown by comparison with the corresponding linear sequence (closed circle minus the end loops) that the effects of end fraying and the strand concentration dependence of the melting temperature are eliminated in the circular form. Over the concentration range examined (0.5 to 2.0 micromolar strands), the circular DNA has a monophasic melting curve, while the linear duplex is biphasic, probably due to hairpin formation. Since effects of duplex to single strands dissociation do not contribute to melting of the circular molecules (dumbells), these DNAs present a realistic experimental model for examining local thermal stability in DNA.

Precise localization of several covalent RNA-RNA cross-link in Escherichia coli 16S RNA.

The RNA-RNA cross-linking reagent N-acetyl-N'-(p-glyoxylyl-benzoyl)cystamine, which reacts via its glyoxal residue with guanines not involved in G X C base pairs, has been used to introduce reversible RNA-RNA cross-links into Escherichia coli 16S rRNA. A two-dimensional gel method has been devised for the separation of the cross-linked oligonucleotides, and the precise location of guanines involved in four of these cross-links has been determined by sequencing the oligonucleotides. One cross-link involves guanosines 1315 and 1360 situated in two hairpin end loops of domain III. The other cross-links involves pairs of guanosine situated within the same hairpin end loops.

Arteriographic evaluation of vascular changes in leprosy.

Two hundred and eighty-four arteriograms were performed in the hands and feet 178 leprosy patients. Narrowing and tortuosity of the digital vessels, especially in the presence of bone absorption, were constant features. Digital tufts revealed signs of ischemia. However, in the presence of infection, hyperemia was a definite observation. Diminution in the caliber of the digital arteries and thinning or absence of the vascular end loops prove that ischemia plays a vital role in the production of various lesions in leprosy.

Infrared evidence for the folded-chain model of trans-1,4-polybutadiene

Abstract The influence of the first-order crystalline transformation at about 60°C on the infrared bands of trans-1,4-polybutadiene yields microscopic information that supports the two-component microcrystalline model of a folded chain having loose and tight end loops at the lamellar surfaces. The temperature dependence of the band parameters of the 908-cm−1 vinyl band indicates that 1,2-(side vinyl) units are not easily taken into the crystalline component, thus forming long loops at the crystal surface that account for the micro-Brownian motion detected in the low-temperature phase. The results argue that infrared band parameters are excellent probes for details of the thermodynamics and morphology of polymers.

Considerations regarding the regulation of gene transcription and messenger translation

The RNA sequences in the region of the coat protein gene of MS2/R17 phage, of the maturation protein gene ofQβ and of a 6s-RNA transcribed from a presumptive gene ofλ were examined. All but five of the 61 amino acid codons are present in these messages and although there is some evidence of a bias in the frequency of utilization of the codons, there is little evidence to suggest that any codons may be forbidden. It is shown that selection has not reduced useless protein synthesis by minimizing the number of out of register AUG's or maximizing the number of out of register terminating codons in messenger RNA. Secondary structures of the RNA's were formulated using a modification of the diagonal method of Tinocoet al. (1971). Of the 12 AUG triplets considered in the sequences only one is a primary attachment site for ribosomes and it is the only one of the 12 that appears in end loops with none of the bases paired. A thirteenth AUG triplet appears at the beginning of the 6s-RNAλ gene. Its occurrence in an end loop free of base pairing suggests that this is a ribosome binding site. It is speculated (1), that transcriptase may recognize its binding site on the DNA by a procedure that is in part the analogue of the protein synthetase recognizing its binding-initiation site, namely that as the DNA strands are separated, a secondary structure forms in the sense strand with a CAT in the end loop; (2), that reverse transcriptase might be useful as a means of keeping multiple copies of a gene identical if it were to correct the sense strand DNA in a DNA-RNA hybrid; and (3), that processing of RNA in the intercistronic region normally occurs in the same manner as translation, namely, moving the RNA through, three nucleotides at a time.

Circulation of Water in the Hammond Distribution System

This paper discusses the Hammond, Illinois, water system, which was installed in 1935 and produced a plant effluent excellent in taste, odor, color, hardness, and sanitary quality. For 24 years, however, red water had occurred at the end loops of the system. The iron content of the raw water had been determined at various times to be 5‐9 ppm with a plant effluent of 0.01‐0.5 ppm. Distribution system samples had shown iron concentrations as large as 4.6 ppm and 7.5 ppm in 1955 and 1956. A number of practices were tried with no real success, before it was decided to try induced velocity, that if economically feasible, would be a decided aid to treatment. Different phases of the problem were studied. Analyses were made of the existing flow pattern, to provide time of travel to various points in the existing system, and of data on relative velocities of flow. It was determined whether it was feasible to recirculate water through the plant for reclarification and thus provide periodic circulation in the dead loops with velocities of 1 fps or 2 fps. Studies were made of an alternate system design in order to obtain minimum time of contact in the mains and still satisfy. The paper discusses system analysis, recirculation analysis, and alternate system design.