Macromolecular Design,Nucleic Acid Junctions,and Crystal Formation

Abstract

The simplest form of macromolecular design involves the ligation of nucleic acids. Recent results on the concatenation of nucleic acid junctions show that these molecules can act as fairly rigid macromolecular valence clusters on the nanometer scale. These clusters can be joined to form closed stick figures in which each edge is double helical DNA or RNA and each vertex is a nucleic acid junction. The geometrical criteria for forming discrete-closed and periodic structures from these components are established. The helicity of each edge limits the possible structures that can be formed.

The formation of a periodic array from nucleic acid junction building blocks is compared with the crystallization of molecular systems. This comparison leads to a new interpretation of the nature of order in the solid state for molecular crystals. The suggestion is made that the structure of a solid molecular system described by the fewest unique orthogonal (Fourier) components is the one which will be entropically favored, since it contains the least information. This is the crystalline state, with a small number of molecules per asymmetric unit. The free energy from the proposed entropie driving force responsible for this behavior is available, in principle, to correct small deviations from ideality in forming covalent crystals from nucleic acid junction components, as well as in non-bonded molecular systems. Nucleic acid junction periodic arrays provide an appropriate vehicle with which to test this interpretation.     

Macromolecular design, nucleic acid junctions, and crystal formation

The simplest form of macromolecular design involves the ligation of nucleic acids. Recent results on the concatenation of nucleic acid junctions show that these molecules can act as fairly rigid macromolecular valence clusters on the nanometer scale. These clusters can be joined to form closed stick figures in which each edge is double helical DNA or RNA and each vertex is a nucleic acid junction. The geometrical criteria for forming discrete-closed and periodic structures from these components are established. The helicity of each edge limits the possible structures that can be formed. The formation of a periodic array from nucleic acid junction building blocks is compared with the crystallization of molecular systems. This comparison leads to a new interpretation of the nature of order in the solid state for molecular crystals. The suggestion is made that the structure of a solid molecular system described by the fewest unique orthogonal (Fourier) components is the one which will be entropically favored, since it contains the least information. This is the crystalline state, with a small number of molecules per asymmetric unit. The free energy from the proposed entropic driving force responsible for this behavior is available, in principle, to correct small deviations from ideality in forming covalent crystals from nucleic acid junction components, as well as in non-bonded molecular systems. Nucleic acid junction periodic arrays provide an appropriate vehicle with which to test this interpretation.     

红胫戟纹蝗痘病毒形态及理化性质研究

红胫戟纹蝗Dociostaurus kraussi是新疆草原优势种蝗虫。1989年首次从新疆玛纳斯红胫戟纹蝗上分离到痘病毒Dociostaurus kraussi ntomopoxvirus(DkEPV),1992年又在新疆巴里坤发现, 自然流行率达23.3%。显微镜观察表明该病毒主要感染脂肪体。病毒球状体为圆球状,直径为2—7μm,大小差异悬殊,病毒粒子砖形或椭圆形,表面呈桑椹结构, 大小平均为144nlnx269nn。病毒DNA具有典型的核酸紫外吸收光谱。根据热变性曲线测得DkEPV—DNA的T_m,值为79.0,(G+C)%为23.7%。病毒DNA经限制性内切酶EcoRI、Bgl IIH和Hind III酶切后,分别得到29、21和18个片段。以λDNA Hind III酶切片段为标准分子量,计算出各酶切片段的分子量为155.45x10⁶、155.69x10⁶和155.40x10⁶D, 由此得出DkEPV—DNA总分子量为55.5x10⁶D。     

The non-virus proteins associated with tobacco mosaic virus

1. Exhaustive fractionation of leaves from tobacco plants systemically infected with TMV has led to the isolation of two non-virus proteins, B3 and B6, and the detection of a third, A4, which do not occur in comparable uninfected plants. 2. Components B3 and B6 have been found consistently in a series of ten extracts from plants grown over an 18 month period in all seasons of the year. It is concluded that these components are as characteristic of the infected plant as TMV itself. 3. As they occur in the initial extracts, the non-virus proteins are of low molecular weight (S₂₀ ca. 3). On treatment, each component tends to form a high molecular weight polymer with an electrophoretic mobility considerably greater than that of the starting material. The high molecular weight derivatives of A4, B3, and B6 have been designated A8, B8, and B7 respectively. There is no evidence that these high molecular weight components occur as such in the infected leaf. 4. The non-virus proteins are free of nucleic acid and are not infectious. They cross-react immunochemically with TMV. 5. Compared with TMV content, the amounts of the non-virus proteins found in infected leaf are relatively small, falling in the range of 10 to 150 micrograms per gm. of tissue.     

Molecular feature of the nerve growth factor in human serum

High molecular weight binding components which bind [¹²⁵I] mouse β nerve growth factor exist in human serum. The binding of β nerve growth factor to the serum components was inhibited at alkaline condition. After gel filtration of human serum on a Sephadex G-150 column at neutral condition, the nerve growth factor-like immunoreactivity was observed in only one peak, differing from the high molecular weight serum components. However, at alkaline condition two peaks with nerve growth factor-like immunoreactivity appeared; one was almost at the position observed at neutral pH, and the other was a new peak eluted approximately to the column volume. these results suggest that there are at least two nerve growth factor-like molecules in human serum and most of the nerve growth factor in the serum exists in a complex form associated with serum components with high molecular weight.     

Stimulation of Cu2+-catalyzed Oxidation of Norepinephrine by Nucleic Acid Components

Oxidation of norepinephrine catalyzed by Cu²⁺ was variously regulated with nucleic acid components. The reaction proceeded by a mechanism of sequential random ordered reaction via formation of the mixed complex of nucleic acid component, Cu²⁺ and aromatic reductone. Using norepinephrine as an aromatic reductone, the promoting activities of nucleic acid components on the oxidation of norepinephrine were compared and the effect of these components to the specific stage of the oxidation process was kinetically investigated. The results indicated that velocity of the oxidation was most remarkably stimulated in the presence of adenine. The velocity was followed by guanine, guanosine monophosphate, cytosine, cytidine, NAD⁺, adenosine, cytidine monophosphate, uridine monophosphate, then adenosine monophosphate in that order. It was also discussed that adenine was the most plausible nucleic acid component which could participate in the in vivo oxidation of norepinephrine, taking into account the concentration of Cu²⁺ and nucleic acid components in living tissues.     

Nucleoprotein Complexes of Yeast and Their Biological Effect on T-Lymphocytes

Complexes of nucleic acids and acid nuclear proteins that are active toward human T-lymphocytes were isolated from cells of baker's yeastSaccharomyces cerevisiae. The conditions of isolation of nucleoprotein complexes by acid extraction followed by microfiltration for concentration of macromolecular components were optimized. Gel filtration and electrophoresis were used to study the composition and molecular weights of components of the preparations obtained. It was shown that the nucleoprotein complex had a molecular weight of 1430 kDa. However, only one zone was determined by electrophoresis of the protein component with a molecular weight of 30 kDa.     

Einbau von 3H-Uracil in die RNA steriler Anzuchten von Utricularia stellaris mit und ohne Rohrzucker

Summary Sucrose has been shown to increase the growth of Utricularia stellaris in axenic culture (Harder and Zemlin, 1967). Comparable cultures of Utricularia were incubated for 3 and 7 hours with tritiated uracil, and then nucleic acids were prepared. With sucrose, the incorporation is about 5 times higher than that with the inorganic salt cultures. After chromatographic separation of the nucleic acids on MAK-columns, different peaks of short time labelled RNA with high specific activity were found. These peaks indicate the presence of two low molecular weight RNA components in the inorganic culture. These peaks also have been found in cultures with sucrose, but in these cultures there is another predominant peak of high molecular weight RNA which is eluted from the column after the greater ribosomal RNA component.     

Isolierung und Charakterisierung schnell markierter,hochmolekularer RNS aus frei suspendierten Calluszellen der Petersilie (Petroselinum sativum)

Summary By culturing of callus tissue originating from root explants of Petroselinum sativum in a synthetic liquid medium under aeration, freely suspended single cells and small clusters consisting of mostly five cells were obtained. The rapidly dividing cells did not exhibit any morphogenesis. Their nucleic acid metabolism was investigated by pulse experiments with ³²P-orthophosphate. Rapidly labelled RNA was prominently found associated with high molecular RNA. During the fractionation of the total nucleic acids on MAK columns it was eluted after the ribosomal RNA components. Its base ratio, however, differed from the latter in that the AMP content was higher than the GMP content. Sucrose gradient centrifugation and polyacrylamide gel electrophoresis resulted in the separation of the ribosomal RNA from the rapidly labelled RNA, thus proving the higher molecular weight of the latter. Based upon the migration in the gel a sedimentation coefficient of approximately 32S was calculated. The possible function of the heavy rapidly labelled RNA component as precursor of ribosomal RNA is discussed.     

Intracellular distribution of low molecular weight RNA in Chironomus tentans

Low molecular weight RNA species are described in isolated nuclear components and cytoplasm of salivary gland cells of Chironomus tentans. In addition to 4S and 5S RNA and RNA in the 4–5S range previously described, at least three other components in the range below 16S are present. RNA, the molecular weight of which was estimated to 2.3 x 10⁵ and designed 10S RNA, can be observed only in nucleoli; other RNA, the molecular weight of which was estimated to 1.3 x 10⁵ and designed 8S RNA, was detected in the chromosomes, the nuclear sap, and the cytoplasm but not in the nucleoli; and a third type of RNA, the molecular weight of which was estimated to 8.5 x 10⁴ and designed 7S RNA, was present in nucleoli, chromosomes, nuclear sap, and cytoplasm. The substituted benzimidazole, 5,6-dichloro-1 (β-D-ribofuranosyl)benzimidazole (DRB), which gives a differential inhibition of the labeling of heterodisperse, mainly high molecular weight RNA in the chromosomes, does not inhibit the labeling of 8S RNA. The relative amounts of label in 8S RNA and 4–5S RNA (including 4S RNA and 5S RNA) in different isolated chromosomes, are distributed in proportion to the chromosomal DNA contents. The 8S RNA as well as the 7S RNA show a relative accumulation in chromosomes and nuclear sap with prolonged incubation time and are in this respect similar to intranuclear low molecular weight RNA species described by previous workers. Our data suggest, however, that these two types of RNA may differ in an important aspect from the previously described types since they are also present in the cytoplasm.     

Altered nucleic acid partitioning during phenol extraction or silica adsorption by guanidinium and potassium salts

Nucleic acids were found to partition into the phenol phase during phenol extraction in the presence of guanidinium at certain concentrations under acidic conditions. The guanidinium-concentration-dependent nucleic acid partitioning patterns were analogous to those of the nucleic acid adsorption/partitioning onto silica mediated by guanidinium, which implied that phenol and silica interact with nucleic acids through similar mechanisms. A competition effect was observed in which the nucleic acids that had partitioned into the phenol phase or onto the silica solid phase could be recovered to the aqueous phases by potassium in a molecular weight–salt concentration-dependent manner (the higher molecular weight nucleic acids needed higher concentrations of potassium to be recovered, and vice versa). Methods were developed based on these findings to isolate total RNA from Escherichia coli. By controlling the concentrations of guanidinium and potassium salts used before phenol extraction or silica adsorption, we can selectively recover total RNA but not the high molecular weight genomic DNA in the aqueous phases. Genomic DNA-free total RNA obtained by our methods is suitable for RT-PCR or other purposes. The methods can also be adapted to isolate small RNAs or RNA in certain molecular weight ranges by changing the salt concentrations used.     

The influence of polyamine-nucleic acid complexes on Fe2+ autoxidation

Summary Polyamines are able to affect Fe²⁺ autoxidation in the presence of suitable low molecular weight phosphorus-containing compounds; the inhibitory effect exerted by polyamines is directly related to their ability to bind phosphorus-containing compounds [1].It is well known that polyamines, as polycations at physiological pH, bind strongly to nucleic acids. In this paper it is shown that polyamines, also in the presence of nucleic acids, inhibit Fe²⁺ autoxidation and thus depress the generation of free oxygen radicals. Most of the nucleic acids tested inhibited Fe²⁺ autoxidation although the concentration which causes half maximal effect differs. Polyamine effect on Fe²⁺ autoxidation varies greatly depending on the single or double stranded nature of the nucleic acid. In the present of single stranded nucleic acids, spermine and spermidine potentiate the inhibition of Fez+ autoxidation by these nucleic acids. A relationship exists between the ability of spermine to interact with single stranded nucleic acids and to inhibit Fe²⁺ autoxidation in their presence. When double stranded nucleic acids are present, polyamines reverse the inhibition of Fee+ autoxidation exerted by these nucleic acids. Molecular mechanisms are proposed to explain these experimental results. The hypothesis that polyamines may inhibit oxidative damage caused to nucleic acids by Fe²⁺ autoxidation, is also discussed.Abbreviations poly [A] polyadenylic acid (5) - poly [C] polycytidylic acid (5) - poly [1] polyinosinic acid (5) - poly [G] polyguanylic acid (5) - poly [A. U] polyadenylic-uridylic acid - poly [A] poly [U] polyadenylic-polyuridylic acid     

Use of diazotized di-iodosulfanilic acid to localize the protein components in sarcoplasmic reticulum membranes.

Evidence is presented which indicates that radioiodine labelled diazotized di-iodosulfanilic acid is a useful nonpermeant probe for the localization of the protein components in the vesicles formed from fragmented skeletal muscle sarcoplasmic reticulum. The data obtained suggest that a high molecular weight (∼172,000 daltons) protein component or components and the (Ca⁺⁺ + Mg⁺⁺)-ATPase are located on or near the external surface of these vesicles and that calsequestrin and related Ca-binding proteins are either buried within the membrane structure or located on the internal surface of the vesicle.     

Contitions for optimal growth of a PSTV-infected potato cell suspension and detection of viroid-complementary longer-than-unit-length RNA in these cells

A suspension culture from potato spindle tuber viroid (PSTV)-infected cells of the wild type potato (Solanum demissum) has been established, which is a suitable model system for studying PSTV replicationin vivo. The conditions for rapid growth of these cells and for permanent extensive viroid biosynthesis within them are described. Biosynthesis of PSTV in the potato cells was demonstrated by³²P-incorporation into nucleic acids and their subsequent electrophoretic analysis on polyacrylamide gels. Under optimum culture conditions the amount of³²P-orthophosphate incorporation into PSTV reached 10% of that incorporated into the 2 M LiCl-soluble cellular RNA. (+)PSTV and its complementary form, i.e. (?)PSTV were identified after their electrophoretic separation on polyacrylamide and agarose gels by molecular hybridization. This analysis revealed the presence of six high molecular weight(?)PSTV species, which are possibly multimers of the unit length(+)PSTV molecule consisting of 359 nucleotides.     

Bacterial Degradation of Dissolved Organic Matter from Two Northern Michigan Streams

This study used high-pressure size exclusion chromatography (HPSEC) to measure the changes in molecular weight distributions of dissolved organic matter (DOM) of two Northern Michigan streams following inoculation with bacterial concentrates from the same locations. During the initial 12 h of the experiment, weight average molecular weight (M _w ) of DOM decreased, as high molecular weight components were lost from solution. After 12 h, the M _w of DOM increased, primarily because of a loss of intermediate to lower molecular weight components. Leucine incorporation showed little or no bacterial metabolism during the first 12 h, but metabolism increased substantially after 12 h. The initial loss of high molecular weight components during the period of little or no bacterial metabolism suggests preferential adsorption of these components to the bacterial surfaces, perhaps followed by metabolism. This suggested interpretation is consistent with previous observations of preferential adsorption of higher molecular weight components to viable but non-metabolizing Bacillus subtilis and to mineral surfaces. The latter loss of lower molecular weight components was most likely due to bacterial metabolism of the DOM, which is consistent with previous observations that lower molecular weight components are more biodegradable. The HPSEC technique uses 254 nm wavelength for detection and focuses primarily on humic- and fulvic-type components rather than low molecular weight organic molecules, such as carbohydrates. Thus, results confirmed that humic/fulvic components are biodegradable, but did not address other DOM components.     

Temperature of acrylamide polymerization and electrophoretic mobilities of nucleic acids.

The electrophoretic mobilities of DNA, ribosomal RNAs, and pulse-labeled RNAs were compared on polyacrylamide gels polymerized at temperatures from 4 to 35°C and subjected to electrophoresis at a fixed temperature. DNA migrated the same distance irrespective of polymerization temperature, the ribosomal RNAs, and the major pulse-labeled species (a putative rRNA precursor) migrated more rapidly in gels polymerized at higher temperatures. The linearity of the migration versus the log of the molecular weight remained for the five rRNA species used, but the extrapolated molecular weight of the putative precursor ranged from 1.8 × 10⁶ to 2.5 × 10⁶ depending on polymerization temperatures. By varying polymerization temperatures, the optimal resolution of various groups of RNA species can be obtained. The results are explained in terms of polymerization temperature effects on gel structure as well as nucleic acid conformation.     

Characterization of Potyvirus Isolates from West African Yams (Dioscorea spp.)

In comparative studies on potyviruses from West African yams (Dioscorea spp.) the following isolates were used: Dioscorea greenbanding mosaic virus (DGMV) and a Nigerian yam virus (YV-N), both isolated from Dioscorea rotundata, and a beet mosaic virus isolate from D. alata (BtMV-Y) formerly designated Dioscorea alata ring mottle virus. Naturally infected D. alata containing very few particles of BtMV-Y, contained primarily particles of a second potyvirus (Dioscorea alata virus, DaV) which could not be transmitted but which was included in these studies wherever possible. The normal lengths of DGMV, YV-N, DaV, and BtMV-Y were 754, 772, 805, and 812 nm, respectively. All viruses induced cytplasmic inclusions of the pinwheel type and laminated aggregates. In addition, the nucleoli of BtMV-Y infected cells contained characteristic electron dense inclusions. The buoyant density of purified DGMV and BtMV-Y in CsCl was 1.336 g/cm³ and 1.321 g/cm³, respectively. The sedimention velocities (S_rel) of DGMV, YV-N, and BtMV-Y were 156, 158, and 162 Srespectively. In SDS-polyacrylamide gel electrophoresis the coat protein of purified DGMV and YV-N all migrated as a single band with an apparent molecular weight of 36 kd. Coat protein of purified DaV showed up to 5 bands with molecular weights of 36 to, 32 kd. Polypeptides of purified BtMV-Y had an estimated molecular weight of 35 kd but those from infected plant extracts had a molecular weight of 36 kd. DGMV, YV-N, and BtMV-Y particles contained a single nucleic acid with an apparent molecular weightof 3.2, 3.2, and 3.1 Md, respectively. Using λ-DNA digested with Hind III as a marker, the molecular weight of DGMV and BtMV-Y nucleic acid was calculated to be 3.6 Md ± 10%. The nucleic acid was determined to be single-stranded RNA by enzymatic digestion and by staining with acridine orange. In serological studies using immunoelectron microscopy (IEM), electro-blot immunoassay (EBIA), and enzyme-linked immunosorbent assay (ELISA), DGMV and YV-N were closely related. Strong serological reactions were also obtained in IEM and EBIA when DGMV and YV-N were tested with antiserum to yam mosaic virus (YMV). Antisera against DGMV, YV-N, and YMV also reacted strongly with DaV antigen. Serological reactions between these viruses and BtMV-Y were usually not found or were weak. A very close serological relationship could be detected between BtMV-Y and beet mosaic virus isolated from beet (BtMV); both isolates were also very similar in host range, symptomatology, and cytopathology.     

Molecular crowding effects on structure and stability of DNA

Living cells contain a variety of biomolecules including nucleic acids, proteins, polysaccharides, and metabolites as well as other soluble and insoluble components. These biomolecules occupy a significant fraction (20-40%) of the cellular volume. The total concentration of biomolecules reaches 400gL(-1), leading to a crowded intracellular environment referred to as molecular crowding. Therefore, an understanding of the effects of molecular crowding conditions on biomolecules is important to broad research fields such as biochemical, medical, and pharmaceutical sciences. In this review, we describe molecular conditions in the cytoplasm and nucleus, which are totally different from in vitro conditions, and then show the biochemical and biophysical consequences of molecular crowding. Finally, we discuss the effect of molecular crowding on the structure, stability, and function of nucleic acids and the significance of molecular crowding in biotechnology and nanotechnology.     

Time course of gold-induced accumulation of copper and zinc and the effects of dimercaptosuccinate and cadmium on gold metabolism in rat kidney

After the s.c. administration of sodium aurothiomalate (SATM, 10 mg Au/kg) to male rats, the gold content of the kidney increased to a maximum after 4 days and thereafter declined slowly. The total copper content of the kidney increased at least until 11 days after SATM treatment but was not simply a function of the renal gold content. Gold and copper accumulated in the metallothionein-like, low molecular weight protein fraction and the initial uptake of gold by this fraction appeared to be related to the accumulation of copper. The Zn²⁺ content of the kidney was initially unchanged but later increased with the additional Zn²⁺ bound predominantly to the non-soluble components. The accumulation of low molecular weight protein-bound copper was not related either to the Zn²⁺ content of this fraction or to the total Zn²⁺ content of the kidney.Daily administration of dimercaptosuccinic acid (DMSA, 50 mg/kg, i.p.) for 2 weeks to SATM-pretreated rats resulted in a 50% reduction in the concentration of gold in the non-soluble fraction and in both the high and low molecular weight protein components of the soluble fraction. Copper, which accumulated in the non-soluble components and in the soluble low molecular weight protein, was lost only from the non-soluble components.Uptake of gold by the kidneys was increased when rats were pretreated with Cd²⁺. The accumulation of gold in the low molecular weight protein fraction was also increased, but most of the additional gold in the kidneys was accumulated by the non-soluble components. SATM had no effect on the renal concentration or subcellular distribution of Cd²⁺. It was concluded that the soluble low molecular weight metal binding protein does not have prominent regulatory or protective functions in the renal metabolism of gold.     

Heparin binding to antithrombin III: Variation in binding sites and affinity

Heparin fractions of different molecular weights and anticoagulant activities were prepared by chromatography on protamine-Sepharose, and the association constants and stoichiometry for binding to antithrombin III were determined by measurement of enhancement of tryptophan fluorescence. A 7,900 molecular weight heparin preparation bound to antithrombin III with a stoichiometry of close to 2:1, whereas 14,300 and 21,600 molecular weight fractions bound at approximately 1:1 with the protein. Apparent association constants were 0.66 × 10⁶ M^?1 for the low molecular weight preparation and 2.89 × 10⁶ M^?1 for the high molecular weight material. Maximal fluorescence enhancement was greater with the higher molecular weight heparin. These results suggest a model of heparin-antithrombin III binding in which two sites on antithrombin III can accommodate one large heparin molecule with high affinity or two smaller molecules with low affinity.