bluuebunny/crossref_metadata_2025 huggingface dataset:
Disparate differentiation in mouse hemopoietic colonies derived from paired progenitors.
Authors: Suda T , Suda J , Ogawa M
Published: May 2006

We analyzed the differentiation of murine hemopoietic colonies derived from paired progenitors in culture. Single progenitors were isolated by use of a micromanipulation technique from blast cell colonies cultured from the spleens of 5-fluorouracil-treated mice. Eighteen to 24 hr later, the paired progenitors were separated with a micromanipulator and cultured in methylcellulose medium containing erythropoietin and pokeweed-mitogen spleen cell conditioned medium. Six to nine days later, the two colonies derived from the paired progenitors were individually picked and differential counts were performed by using May-Grunwald-Giemsa stain. The abbreviations used here are n, neutrophil; m, macrophage; e, eosinophil; mast, mast cell; M, megakaryocyte; E, erythrocyte. Of a total of 387 pairs that could be evaluated, 68 were pairs of colonies consisting of dissimilar combinations of cell lineages such as m-nmmastEM, M-nmmastEM, nm-nmmastEM, nmmastM-nmmastEM, M-nmmastM, nmmast-nmmastM, nm-nmmastE, M-nmM, n-nmM, mM-nmM, m-nmmast, nm-nme, me-nm, mM-nm, n-ne, m-mmast, m-mM, M-nm, M-mM, E-nm, m-nm, M-m, etc. Thirty-nine were homologous pairs revealing identical lineage combinations such as nmmastEM, nmmastM, nmmast, mmastEM, nmEM, nme, nmM, mM, and nm lineages. However, in members of some of these pairs, the proportions of the individual cell lineages were significantly different. The remainder were pairs of single lineage colonies. Paired progenitors obtained from the stem cell colonies of normal mice also revealed homologous and nonhomologous expression of the cell lineages. Comparison of lineage expression in colonies derived from single progenitors with the sum of lineages expressed in pairs of colonies derived from single progenitors indicated that the diversity was not due to injury inflicted by micromanipulation. These observations provide experimental data in support of stochastic mechanisms of stem cell differentiation.
Histamine-containing neurons in the rat hypothalamus.
Authors: Panula P , Yang H Y , Costa E
Published: May 2006

A specific antiserum against histamine was produced in rabbits, and an immunohistochemical study of histamine-containing cells was carried out in rat brain. The antiserum bound histamine in a standard radioimmunoassay and stained mast cells located in various rat and guinea pig tissues. Enterochromaffin-like cells in the stomach and neurons in the posterior hypothalamic area could be detected with this antiserum. The staining was highly specific and was not abolished by preabsorption with histidine, histidine-containing peptides, serotonin, or catecholamines, whereas preabsorption with histamine completely abolished the staining. Immunoglobulins of this antiserum purified by affinity chromatography stained the same cells as did the crude antiserum, whereas the serum fraction, which was not absorbed by histamine-affinity ligand, failed to stain any neuron. Histamine-immunoreactive neuronal cell bodies were found only in the hypothalamic and premammillary areas of colchicine-treated rats. The largest group of cells was seen in the caudal magnocellular nucleus and medially on the dorsal and ventral aspects of the ventral premammillary nucleus. Immunoreactive nerve fibers, but no cell bodies, were detected in other parts of the brain. Histamine-immunoreactive mast cells were found in the median eminence and pituitary gland. The results suggest that histamine-containing neurons are located only in a small area of the posterior hypothalamus, and these cells are probably the source of ascending and descending fibers detected in other brain areas.
Charge movement in skeletal muscle fibers paralyzed by the calcium-entry blocker D600.
Authors: Hui C S , Milton R L , Eisenberg R S
Published: May 2006

We report measurements of nonlinear charge movement in frog skeletal muscle fibers paralyzed by the calcium-entry blocker [Schwartz, A. & Taira, N., eds. (1983) Circ. Res. 52, Part II, Number 2, 1-181.] D600 (methoxyverapamil, recently renamed gallopamil). Nonlinear charge movement was not seen in such fibers, suggesting that the drug severs the link between membrane depolarization and the main components of charge movement. This is the only pharmacological agent that blocks the main components of charge movement.
Relationship between lateral diffusion, collision frequency, and electron transfer of mitochondrial inner membrane oxidation-reduction components.
Authors: Gupte S , Wu E S , Hoechli L , Hoechli M , Jacobson K , Sowers A E , Hackenbrock C R
Published: May 2006

Fluorescence recovery after photobleaching was used to determine the diffusion coefficients of the oxidation-reduction (redox) components ubiquinone, complex III (cytochromes b-c1), cytochrome c, and complex IV (cytochrome oxidase) of the mitochondrial inner membrane. All redox components diffuse in two dimensions as common-pool electron carriers. Cytochrome c diffuses in two and three dimensions concomitantly, and its diffusion rate, unlike that of all other redox components, is modulated along with its activity by ionic strength. The diffusion coefficients established in this study reveal that the theoretical diffusion-controlled collision frequencies of all redox components are greater than their experimental maximum (uncoupled) turnover numbers. Since electron transport is slower than the theoretical limit set by the lateral diffusion of the redox components, ordered chains, assemblies, or aggregates of redox components are not necessary to account for electron transport. Rather, mitochondrial electron transport is diffusion coupled, consistent with a "random-collision model" for electron transport.
Two-dimensional S1 nuclease heteroduplex mapping: detection of rearrangements in bacterial genomes.
Authors: Yee T , Inouye M
Published: May 2006

A method of two-dimensional S1 nuclease heteroduplex mapping was developed to detect gene rearrangements and repeated sequences in total bacterial chromosomes. To detect DNA rearrangements between two variant bacterial strains, total chromosomal DNA preparations from the two strains are digested with four-base-recognizing restriction enzymes, mixed together, denatured, renatured, and separated on first-dimension polyacrylamide slab gels. Gel strips are cut out and soaked in a buffer containing S1 nuclease, which diffuses into the strips and digests the DNA fragments at single-stranded regions. The digested DNA is then electrophoresed in a second dimension perpendicular to the first dimension. DNA heteroduplexes that were digested by the S1 nuclease are resolved as distinct spots below a bright unresolved band of homoduplex. This report describes testing of this method on a model system consisting of two nearly isogeneic strains of Escherichia coli, and the application of this method in detecting DNA rearrangements associated with phase variation in Myxococcus xanthus.
Molecular cloning of region-specific chorion-encoding RNA sequences.
Authors: Regier J C , Hatzopoulos A K , Durot A C
Published: May 2006

We have constructed a cDNA clone library from poly(A)+ RNA of very-late-period choriogenic silkmoth follicles. Clone DNAs that hybridize preferentially to RNA from the aeropyle crown region of the follicle (versus the flat region) were selected, and all could be placed in one of two homology groups. The two groups represent sequences encoding the very-late-period chorion proteins E1 and E2; this was established by hybrid-selected translation coupled with specific antibody precipitation. Regionalized synthesis of chorion proteins is restricted to the very late period, and its control can now be studied at the nucleic acid level.
Predicted secondary structure similarity in the absence of primary amino acid sequence homology: hepatitis B virus open reading frames.
Authors: Schaeffer E , Sninsky J J
Published: May 2006

Proteins that are related evolutionarily may have diverged at the level of primary amino acid sequence while maintaining similar secondary structures. Computer analysis has been used to compare the open reading frames of the hepatitis B virus to those of the woodchuck hepatitis virus at the level of amino acid sequence, and to predict the relative hydrophilic character and the secondary structure of putative polypeptides. Similarity is seen at the levels of relative hydrophilicity and secondary structure, in the absence of sequence homology. These data reinforce the proposal that these open reading frames encode viral proteins. Computer analysis of this type can be more generally used to establish structural similarities between proteins that do not share obvious sequence homology as well as to assess whether an open reading frame is fortuitous or codes for a protein.
Biological system interactions.
Authors: Adomian G , Adomian G E , Bellman R E
Published: May 2006

Mathematical modeling of cellular population growth, interconnected subsystems of the body, blood flow, and numerous other complex biological systems problems involves nonlinearities and generally randomness as well. Such problems have been dealt with by mathematical methods often changing the actual model to make it tractable. The method presented in this paper (and referenced works) allows much more physically realistic solutions.
Immunophotoelectron microscopy: the electron optical analog of immunofluorescence microscopy.
Authors: Birrell G B , Habliston D L , Nadakavukaren K K , Griffith O H
Published: May 2006

The electron optical analog of immunofluorescence microscopy combines three developments: (i) photo-electron microscopy to produce a high-resolution image of exposed components of the cell, (ii) site-specific antibodies, and (iii) photoemissive markers coupled to the antibodies to make the distribution of sites visible. This approach, in theory, provides a way to extend the useful immunofluorescence microscopy technique to problems requiring much higher resolution. The resolution limit of fluorescence microscopy is limited to about 200 nm by the wavelength of the light used to form the image, whereas in photoelectron microscopy the image is formed by electrons (current resolution: 10-20 nm; theoretical limit: 5 nm or better depending on the electron optics). As a test system, cytoskeletons of CV-1 epithelial cells were prepared under conditions that preserve microtubules, and the microtubule networks were visualized by both indirect immunofluorescence and immunophotoelectron microscopy using colloidal gold coated with antibodies. Colloidal gold serves as a label for immunophotoelectron microscopy, providing enhanced photoemission from labeled cellular components so that they stand out against the darker background of the remaining unlabeled structures. In samples prepared for both immunofluorescence and immunophotoelectron microscopy, individual microtubules in the same cells were visualized by both techniques. The photoemission of the colloidal gold markers is sufficiently high that the microtubules are easily recognized without reference to the immunofluorescence micrographs, indicating that this approach can be used, in combination with antibodies, to correlate structure and function in cell biological studies.
Mechanism of activation of the heme-stabilized translational inhibitor of reticulocyte lysates by calcium ions and phospholipid.
Authors: de Herreros A G , de Haro C , Ochoa S
Published: May 2006

We have reported previously that calcium ions and phospholipid activate the heme-stabilized proinhibitor form (pro-HCI) of the heme-controlled translational inhibitor (HCI) in reticulocyte lysates and promote the first step of the reaction pro-HCI in equilibrium reversible HCI----irreversible HCI. This suggested the possible involvement of a Ca2+/phospholipid-dependent protein kinase (protein kinase C) in the activation. However, further investigation revealed, among other things, that polyunsaturated fatty acids (e.g., arachidonic acid) were as effective as Ca2+/phospholipid in promoting translational inhibition and phosphorylation of the alpha subunit of the chain-initiation factor eIF-2 and, moreover, HCI activation could be prevented or reversed in either case by NADPH-generating systems or by dithiols. Our results suggest that pro-HCI is activated by lipoperoxides produced in reticulocyte lysates from either phospholipid or polyunsaturated fatty acids; the presence of Ca2+ is required in the former but not in the latter case. The reversible activation of HCI by Ca2+ and phospholipid might suggest a possible modulatory role of Ca2+ in translational control.
Cell-type-specific receptors for alpha-fetoprotein in a mouse T-lymphoma cell line.
Authors: Naval J , Villacampa M J , Goguel A F , Uriel J
Published: May 2006

Binding and uptake of alpha-fetoprotein (AFP) by mouse T-lymphoma YAC-1 cells exhibited the characteristics of receptor-mediated endocytosis. The binding saturation curve obtained by incubating YAC-1 cells at 4 degrees C with 125I-labeled AFP at different concentrations (50 ng/ml to 2.5 mg/ml) showed three saturation plateaus. AFP binding was inhibited by unlabeled mouse, rat, or bovine AFP and, to a lesser extent, by rat or bovine serum albumin. No significant competition was observed with transferrin, alpha 2-macroglobulin, IgG, or ovalbumin. Scatchard analysis suggested the presence of three types of receptor sites with a Kd of 2.2 X 10(-9) M (approximately equal to 700 sites per cell), 8.6 X 10(-7) M (approximately equal to 210,000 sites per cell), and 5.7 X 10(-6) M (approximately equal to 910,000 sites per cell), respectively. At 37 degrees C, AFP was rapidly internalized and could be localized in the cytoplasm after incubation of cells with fluoresceinated AFP. After a short residence time, AFP was released undegraded from the cells. Normal adult thymocytes and T lymphocytes, which are counterparts of YAC-1 cells, did not show any significant uptake of AFP. On the other hand, a small subpopulation of fetal and newborn thymocytes was labeled by fluoresceinated AFP.
A systematic study of HLA class II-beta DNA restriction fragments in insulin-dependent diabetes mellitus.
Authors: Cohen-Haguenauer O , Robbins E , Massart C , Busson M , Deschamps I , Hors J , Lalouel J M , Dausset J , Cohen D
Published: May 2006

DNA restriction fragments of the genes encoding HLA class II-beta antigens were compared in 34 patients with insulin-dependent diabetes mellitus and 34 HLA-DR-matched healthy individuals. Ninety-three fragments, determined by six restriction enzymes (EcoRI, EcoRV, HindIII, BamHI, Pvu II, and Taq I), were analyzed: (i) A DR Taq I 12.7-kilobase-pair fragment might be a marker for the extended haplotype HLA-B8, DR3. (ii) In controls, DR4 haplotypes are associated with two distinct clusters of DQ restriction fragments (DQR4 and DQR5). Almost all (94%) DR4 patients belong to the DQR4 and not to the DQR5 cluster. This suggests that, among HLA-DR4 haplotypes, only DQR4 haplotypes are involved in susceptibility to insulin-dependent diabetes mellitus. (iii) A DR Taq I 14.5-kilobase-pair fragment was found to be strongly associated with DQR4, mainly in DR3/DR4 heterozygous patients (P = 5 X 10(-4). However, these results must be interpreted with caution, taking into account the high number of statistical tests performed.
Relative rates of homologous and nonhomologous recombination in transfected DNA.
Authors: Roth D B , Wilson J H
Published: May 2006

Both homologous and nonhomologous recombination events occur at high efficiency in DNA molecules transfected into mammalian cells. Both types of recombination occur with similar overall efficiencies, as measured by an endpoint assay, but their relative rates are unknown. In this communication, we measure the relative rates of homologous and nonhomologous recombination in DNA transfected into monkey cells. This measurement is made by using a linear simian virus 40 genome that contains a 131-base-pair duplication at its termini. Once inside the cell, this molecule must circularize to initiate lytic infection. Circularization can occur either by direct, nonhomologous end-joining or by homologous recombination within the duplicated region. Although the products of the two recombination pathways are different, they are equally infectious. Since homologous and nonhomologous recombination processes are competing for the same substrate, the relative amounts of the products of each pathway should reflect the relative rates of homologous and nonhomologous recombination. Analysis of individual recombinant genomes from 164 plaques indicates that the rate of circularization by nonhomologous recombination is 2- to 3-fold higher than the rate of homologous recombination. The assay system described here may prove to be useful for testing procedures designed to influence the relative rates of homologous and nonhomologous recombination.
Focal smooth muscle proliferation in the aortic intima produced by an initiation-promotion sequence.
Authors: Majesky M W , Reidy M A , Benditt E P , Juchau M R
Published: May 2006

Human atherosclerotic fibrous plaques display a clonal character similar to many benign neoplasms. We report here that chickens treated with an initiation-promotion sequence developed focal intimal smooth muscle lesions in the thoracic aorta that resemble early forms of atherosclerosis. Scanning electron microscopy revealed small mound-like lesions protruding from an intact endothelium in birds treated with an initiating dose of 7,12-dimethylbenz[a]anthracene (Me2BA) followed by twice weekly injections of the alpha 1-selective adrenergic agonist methoxamine for 20 weeks. Intimal lesion foci were composed of densely packed modified smooth muscle cells, abundant extracellular matrix, and occasional mononuclear cells (possibly monocytes). There was no ultrastructural evidence of lipid accumulation or alteration of the underlying media. These intimal lesions appeared in aortic segments of treated chickens in a pattern similar to that observed in classical experiments of multistage tumorigenesis in epidermis and other tissues. The treatment with Me2BA followed by methoxamine produced more focal lesions per thoracic segment and more segments per group with lesions than did treatment with either Me2BA or methoxamine alone. Thoracic intimal foci were absent from untreated and vehicle-treated groups. In contrast, the growth of a spontaneously arising atheroma in the distal abdominal aorta was not demonstrably affected by the initiation-promotion regimen. Likewise, weekly injections of Me2BA for 23 weeks, while greatly enhancing abdominal atheroma growth, produced no thoracic lesions. These results provide evidence that focal proliferation of intimal smooth muscle cells, a critical early event in atherogenesis, can be produced by an initiation-promotion treatment sequence.
Batrachotoxin changes the properties of the muscarinic receptor in rat brain and heart: possible interaction(s) between muscarinic receptors and sodium channels.
Authors: Cohen-Armon M , Kloog Y , Henis Y I , Sokolovsky M
Published: May 2006

The effects of Na+-channel activator batrachotoxin (BTX) on the binding properties of muscarinic receptors in homogenates of rat brain and heart were studied. BTX enhanced the affinity for the binding of the agonists carbamoylcholine and acetylcholine to the muscarinic receptors in brainstem and ventricle, but not in the cerebral cortex. Analysis of the data according to a two-site model for agonist binding indicated that the effect of BTX was to increase the affinity of the agonists to the high-affinity site. Guanyl nucleotides, known to induce interconversion of high-affinity agonist binding sites to the low-affinity state, canceled the effect of BTX on carbamoylcholine and acetylcholine binding. BTX had no effect on the binding of the agonist oxotremorine or on the binding of the antagonist [3H]-N-methyl-4-piperidyl benzilate. The local anesthetics dibucaine and tetracaine antagonized the effect of BTX on the binding of muscarinic agonists at concentrations known to inhibit the activation of Na+ channels by BTX. On the basis of these findings, we propose that in specific tissues the muscarinic receptors may interact with the BTX binding site (Na+ channels).
Initiation of enzymatic replication at the origin of the Escherichia coli chromosome: contributions of RNA polymerase and primase.
Authors: Ogawa T , Baker T A , van der Ende A , Kornberg A
Published: May 2006

Replication of plasmids that depend on the 245-base-pair origin of the Escherichia coli chromosome (oriC) requires many purified proteins that (i) direct initiation to oriC (e.g., dnaA protein), (ii) influence initiations elsewhere (e.g., auxiliary proteins), and (iii) prime and extend DNA chains (e.g., priming and synthesis proteins). For the RNA priming and initiation of new DNA chains, the requirements for both primase and RNA polymerase (RNA pol) [Kaguni, J. M. & Kornberg, A. (1984) Cell 38, 183-190] have been further analyzed. Depending on the levels of auxiliary proteins (topoisomerase I and protein HU), three priming systems can operate: primase alone, RNA pol alone, or both combined. At low levels of auxiliary proteins, primase alone sustains an effective priming system. At higher levels, primase action is blocked, but RNA pol alone can initiate replication, albeit feebly; at these high levels of auxiliary proteins, primase and RNA pol act synergistically. When RNA pol is stalled by an inhibitor or lack of a ribonucleoside triphosphate, primase action is also inhibited. Based on these and other data [van der Ende, A., Baker, T. A., Ogawa, T. & Kornberg, A. (1985) Proc. Natl. Acad. Sci. USA 82, in press], RNA pol can counteract inhibition by auxiliary proteins and thus activate the origin for the priming by primase of the leading strand of the replication fork.
Characterization of rate-controlling steps in vivo by use of an adjustable expression vector.
Authors: Walsh K , Koshland D E
Published: May 2006

Citrate synthase (EC 4.1.3.7) was varied from 10% to 5000% the level found in wild-type Escherichia coli by means of recombinant DNA techniques. When acetate was the sole carbon source, cell growth and carbon flow through the Krebs cycle were greatly affected by the under-production of citrate synthase. In contrast, when glucose was the main nutrient, the same underproduction of citrate synthase had little effect on either growth or carbon flux. When the enzyme was overproduced 50-fold, cultures would grow on glucose but cell division could be abruptly stopped by adding acetate to the medium. These results indicate that the regulatory properties of citrate synthase are highly dependent on the carbon-source composition of the medium. Furthermore, recombinant DNA technology can be used to alter rate-controlling steps in biological pathways and elucidate the regulatory properties of metabolic systems.
Crystal structure of Z-DNA without an alternating purine-pyrimidine sequence.
Authors: Wang A H , Gessner R V , van der Marel G A , van Boom J H , Rich A
Published: May 2006

In left-handed Z-DNA, consecutive nucleotides along the chain alternate in the syn and anti conformations. Purine residues form the syn conformation readily and up to now all Z-DNA crystal structures have sequences of alternating purines and pyrimidines. However, we find that d(C-G-A-T-C-G) with the cytosines brominated or methylated on C-5 crystallizes as Z-DNA. The structure reveals thymines in syn and adenines in anti conformations. This suggests that Z-DNA may occur in sequences other than those with alternating purine-pyrimidine sequence.
A model of protein conformational substates
Authors: Stein D. L.
Published: May 2006

Many proteins have been observed to exist in a large number of conformations that are believed to play an important role in their dynamics. A model of protein conformational substates that incorporates the ideas of frustration and disorder in analogy to glasses and spin glasses is proposed. Applications to x-ray diffraction, Mössbauer studies, and recombination experiments are discussed.
Supramolecular assemblies of mRNA direct the coordinated synthesis of type I procollagen chains.
Authors: Veis A , Leibovich S J , Evans J , Kirk T Z
Published: May 2006

Registration of the three procollagen alpha chains and assembly of the triple-helical procollagen molecules takes place in the rough endoplasmic reticulum, but the exact location and timing of assembly is not known. As part of a study of the mechanism of molecular assembly, intact collagen-producing polyribosomes from embryonic chicken tendon fibroblasts have been examined by the techniques of rotary shadowing and electron microscopy. Intact mRNA strands corresponding in length to approximately 4500 bases and complete procollagen alpha (I) chains have been observed. The mRNA strands are comprised of two mRNA chains. The ribosomes are present in pairs separated along the duplex strand by about 100 nm. The intact polysome is asymmetric; two duplex strands join, and large ribosome aggregates appear. These aggregates are dispersed by collagenase digestion, leaving separate duplex strands with ribosome pairs intact. Ribonuclease digestion yields mixtures of monosomes and ribosome aggregates. Sequential ribonuclease and collagenase digestions yield only monosomes. We propose that each ribosome reads one mRNA chain, so that each pair is thus translating two chains in synchrony. Thus, the complex morphology of the collagen-producing polyribosomes suggests that the organization of a single molecule begins by the organization of the mRNA chains themselves.
Centipedal hemocyanin: its structure and its implications for arthropod phylogeny.
Authors: Mangum C P , Scott J L , Black R E , Miller K I , Van Holde K E
Published: May 2006

The oxygen carrier hemocyanin occurs in the blood of Scutigera coleoptrata, a uniramous arthropod, as well as the crustaceans and chelicerates. The native polymer appears to be composed of substructures having the same size and electron-dense image as those of other arthropod hemocyanins but assembled into a unique multiple and arranged in a unique configuration. The simplest explanation of these findings is that the arthropod hemocyanins have a common origin, exemplifying a derived (as opposed to primitive) character shared by each of the three living groups.
Membrane knobs are required for the microcirculatory obstruction induced by Plasmodium falciparum-infected erythrocytes.
Authors: Raventos-Suarez C , Kaul D K , Macaluso F , Nagel R L
Published: May 2006

We have studied the pathophysiology of the vascular obstruction induced by Plasmodium falciparum-parasitized erythrocytes with the use of an ex vivo microcirculatory preparation perfused with red cells infected with knobless and knobby clones of the FCR-3 strain. We find that parasitized erythrocyte membrane knobs are indispensable for the generation of the circulatory obstruction. Uninfected erythrocytes incubated in culture and erythrocytes infected with early or late forms of the knobless clones or the early forms of the knobby clone all failed to obstruct the microcirculation, although exhibiting various effects on bulk viscosity and peripheral resistance during flow. In contrast, late forms of the knobby clone produced significantly higher peripheral resistance during flow and significant obstruction as detected by changes in time of pressure flow recovery as well as by direct videorecorded microscopic observation. Optical and electron microscopy showed that the adherence of parasitized cells to the endothelium was limited to the venules and involved the knobs in junctions. In addition, we were able to follow the sequence of events during obstruction: initial red-cell adherence to the venular endothelium (sometimes only transitory) followed by progressive recruitment at the venule surface, finally leading to total obstruction that involved parasitized and nonparasitized erythrocytes. Sometimes, retrograde aggregation would extend the obstruction to the capillaries or even precapillary arterioles. These results show that knobs are necessary and sufficient to produce vascular obstruction and that other factors (spleen, immunological, etc.) can only have a modulating role. These results also exclude the possibility that the exclusive adherence to venules is the consequence of "plasma factors" found in the malaric patients.
Initiation of enzymatic replication at the origin of the Escherichia coli chromosome: primase as the sole priming enzyme.
Authors: van der Ende A , Baker T A , Ogawa T , Kornberg A
Published: May 2006

The enzymatic replication of plasmids containing the unique (245 base pair) origin of the Escherichia coli chromosome (oriC) can be initiated with any of three enzyme priming systems: primase alone, RNA polymerase alone, or both combined (Ogawa, T., Baker, T. A., van der Ende, A. & Kornberg, A. (1985) Proc. Natl. Acad. Sci. USA 82, 3562-3566). At certain levels of auxiliary proteins (topoisomerase I, protein HU, and RNase H), the solo primase system is efficient and responsible for priming synthesis of all DNA strands. Replication of oriC plasmids is here separated into four stages: (i) formation of an isolable, prepriming complex requiring oriC, dnaA protein, dnaB protein, dnaC protein, gyrase, single-strand binding protein, and ATP; (ii) formation of a primed template by primase; (iii) rapid, semiconservative replication by DNA polymerase III holoenzyme; and (iv) conversion of nearly completed daughter molecules to larger DNA forms. Optimal initiation of the leading strand of DNA synthesis, over a range of levels of auxiliary proteins, appears to depend on transcriptional activation of the oriC region by RNA polymerase prior to priming by primase.
Significance of similarities in patterns: an application to beta interferon-related DNA on human chromosome 2.
Authors: May L T , Landsberger F R , Inouye M , Sehgal P B
Published: May 2006

The nucleotide sequence of a 14-kilobase (kb) region of the human beta interferon (IFN-beta)-related DNA locus on chromosome 2 (genomic DNA clone lambda B3) was determined and compared to that of the IFN-beta 1 gene by using the Sellers TT algorithm. This algorithm aligns segments of one sequence with similar segments in a second sequence. A strategy was developed for assessing the significance of similarities between DNA sequences based on a scheme that recognizes patterns or runs of identities within an alignment. The pattern score (II) thus obtained is an entropy-like measure. Numerically it is a reflection of the length of the second longest run of identity in an alignment plus a correction factor due to the other shorter identity runs in the alignment. When the IFN-beta 1 gene is compared to a random nucleotide sequence, the distribution of II scores in such comparisons fits a Gaussian function. This strategy has been used to identify seven segments along one strand of lambda B3 DNA that are related to segments in IFN-beta 1; these seven alignments have II scores greater than or equal to 3 standard deviations above the mean score obtained in comparisons between IFN-beta 1 and random nucleotide sequences. One of these alignments (section 7) has a II score 8.02 standard deviations above this mean score. The likelihood of finding an alignment statement as good as that in section 7 in a random sequence the length of the human genome is approximately 10(-7). Furthermore, the lambda B3 DNA sequence in section 7 selects the human IFN-beta 1 gene as the most significant alignment in computer searches of mammalian nucleotide sequence data bases.
Interactions between acidic proteins and crystals: stereochemical requirements in biomineralization.
Authors: Addadi L , Weiner S
Published: May 2006

Acidic matrix macromolecules are intimately involved in biological crystal growth. In vitro experiments, in which crystals of calcium dicarboxylate salts were grown in the presence of aspartic acid-rich proteins, revealed a stereochemical property common to all the interacting faces. Calcite crystals are nucleated on stereochemically analogous faces when proteins are adsorbed onto a rigid substrate. The importance of this property in biomineralization is discussed.
Nodulin-24 gene of soybean codes for a peptide of the peribacteroid membrane and was generated by tandem duplication of a sequence resembling an insertion element
Authors: Katinakis Panagiotis , Verma Desh Pal S.
Published: May 2006

A nodulin gene coding for a polypeptide with an apparent
M
r
of 24,000 (nodulin-24) was isolated from soybean (
Glycine max
). DNA sequence analysis of this gene revealed that its coding capacity is for a polypeptide of only
M
r
15,100 and is interrupted by four introns. The three middle exons and their flanking segments appear to have been generated by duplications of a unit resembling an insertion sequence. This unit is bounded by a 12-base-pair inverted repeat and encompasses the 54-base-pair exon corresponding to each of three central hydrophobic domains of the protein, nodulin-24. The resulting repeated hydrophobic structure of this protein may be responsible for an apparent increase in
M
r
from 15,100 to 24,000.
In vitro
translation and immunological studies suggest that nodulin-24 is a precursor and is processed cotranslationally into a
M
r
20,000 polypeptide. This polypeptide is a component of the membrane envelope enclosing the bacteroids (peribacteroid membrane) synthesized during symbiosis with
Rhizobium
. The low degree (<6%) of sequence divergence among the repeated units suggests that this gene has been generated recently during the evolution of symbiotic nitrogen fixation in soybean.
Differences in mutagenic and recombinational DNA repair in enterobacteria.
Authors: Sedgwick S G , Goodwin P A
Published: May 2006

The incidence of recombinational DNA repair and inducible mutagenic DNA repair has been examined in Escherichia coli and 11 related species of enterobacteria. Recombinational repair was found to be a common feature of the DNA repair repertoire of at least 6 genera of enterobacteria. This conclusion is based on observations of (i) damage-induced synthesis of RecA-like proteins, (ii) nucleotide hybridization between E. coli recA sequences and some chromosomal DNAs, and (iii) recA-negative complementation by plasmids showing SOS-inducible expression of truncated E. coli recA genes. The mechanism of DNA damage-induced gene expression is therefore sufficiently conserved to allow non-E. coli regulatory elements to govern expression of these cloned truncated E. coli recA genes. In contrast, the process of mutagenic repair, which uses umuC+ umuD+ gene products in E. coli, appeared less widespread. Little ultraviolet light-induced mutagenesis to rifampicin resistance was detected outside the genus Escherichia, and even within the genus induced mutagenesis was detected in only 3 out of 6 species. Nucleotide hybridization showed that sequences like the E. coli umuCD+ gene are not found in these poorly mutable organisms. Evolutionary questions raised by the sporadic incidence of inducible mutagenic repair are discussed.
Molecular basis of mutations at the waxy locus of maize: correlation with the fine structure genetic map.
Authors: Wessler S R , Varagona M J
Published: May 2006

More than 40 mutant alleles of the waxy (Wx) locus of maize are available for molecular analysis. Previous studies have examined the nature of phenotypically unstable Wx mutant alleles caused by insertion of the maize transposable activator (Ac) and dissociation (Ds) elements. In this study we have used Southern blot analysis to characterize the locus in 22 strains harboring wx alleles with stable mutant phenotypes. Of these mutations, 17 are of spontaneous origin, 4 were induced by gamma rays, and 1 was induced by ethyl methanesulfonate. Of these 22 alleles, we find that 13 have either insertions or deletions within the Wx transcription unit. The insertions range in size from 150 base pairs to 6.1 kilobases. For 4 of the 6 deletions identified, the two breakpoints are within the Wx gene. For 9 other alleles we can detect no obvious lesions within or around the transcription unit. Evidence is presented that the insertions and deletions result in the mutant phenotype and are not polymorphisms. This conclusion is based on two findings: (i) a survey of inbred lines revealed only a single instance of polymorphism within the transcription unit, whereas all of the lesions described alter the transcription unit; and (ii) there is an excellent correlation between the position of these lesions on the physical map and their relative position on a fine structure genetic map of the locus.
Amino acid sequence of the alpha subunit of transducin deduced from the cDNA sequence.
Authors: Medynski D C , Sullivan K , Smith D , Van Dop C , Chang F H , Fung B K , Seeburg P H , Bourne H R
Published: May 2006

Transducin, a GTP-binding protein involved in phototransduction in the vertebrate retina, belongs to a family of homologous coupling proteins that also includes Gs and Gi, the regulatory proteins of adenylate cyclase. Here we report the cDNA sequence and deduced amino acid sequence of transducin's alpha subunit (T alpha). The cDNA was isolated, by screening with an antibody probe, from a bovine retinal cDNA library in the expression vector lambda gt11. The 2.2-kilobase cDNA insert hybridized to a single 2.6-kilobase poly(A)+ RNA species present in extracts of bovine retina but not of bovine heart, liver, or brain. The nucleotide sequence of the cDNA revealed an open reading frame long enough to encode the entire 39-kDa T alpha polypeptide. The polypeptide sequence deduced from the cDNA would be composed of 350 amino acids and have a molecular weight of 39,971. Portions of the sequence matched reported amino acid sequences of T alpha tryptic fragments, including sites specifically ADP-ribosylated by cholera and pertussis toxins. The predicted sequence also includes four segments, ranging from 11 to 19 residues in length, that exhibit significant homology to sequences of GTP-binding proteins, including the ras proteins of man and yeast and the elongation factors of ribosomal protein synthesis in bacteria, EF-G and EF-Tu. In combination with previous functional studies of tryptic fragments of T alpha, the deduced amino acid sequence makes it possible to predict which portions of the polypeptide interact with other molecules involved in retinal phototransduction.
Expression of a functional influenza viral cap-recognizing protein by using a bovine papilloma virus vector.
Authors: Braam-Markson J , Jaudon C , Krug R M
Published: May 2006

The gene for the influenza viral PB2 protein, which recognizes and binds the 5'-terminal cap 1 structures (m7GpppNm) on eukaryotic mRNAs, was inserted into a bovine papilloma virus vector under the control of a mouse metallothionein I (MT-I) promoter. After transfection of this vector into mouse NIH 3T3 cells, a cell line, cPB2, was obtained that produces PB2-specific mRNA and authentic PB2 protein. Induction of the MT-I promoter with CdCl2 causes about a 10-fold increase in PB2 mRNA and protein levels. The expressed PB2 protein is functional, as it relieves the block in viral mRNA synthesis exhibited by a temperature-sensitive viral mutant containing a cap-binding defect in the PB2 protein. This demonstrates complementation of a function of a negative-strand RNA virus by a gene product expressed in a cell line from recombinant DNA.
Structural analysis of the umu operon required for inducible mutagenesis in Escherichia coli.
Authors: Kitagawa Y , Akaboshi E , Shinagawa H , Horii T , Ogawa H , Kato T
Published: May 2006

The nucleotide sequence of a 2.8-kilobase fragment of the Escherichia coli chromosome containing the umuDC genes has been determined. The DNA sequence specifies two open reading frames of 417 and 1266 nucleotides encoding proteins with calculated molecular weights of 15,063 and 47,677, respectively. From these and the previous results of genetic and biochemical studies on the cloned genes, we conclude that the former is the umuD and the latter is the umuC gene. In vitro transcription of the regulatory region of the umu operon revealed that (i) upstream of the coding region there is a promoter-operator complex having the consensus sequence, CTGTATATAAAAACAG, of an SOS box, (ii) transcription of the umu operon begins at an adenine residue in the SOS box, and (iii) the LexA protein binds to the operator region, with an equilibrium dissociation constant (Kd) of 0.2, thereby repressing the transcription of the umuDC genes. These results indicate that the expression of the umu operon is under the coordinated control of the recA lexA gene products.
Mitochondrial origins.
Authors: Yang D , Oyaizu Y , Oyaizu H , Olsen G J , Woese C R
Published: May 2006

The 16S ribosomal RNA sequences from Agrobacterium tumefaciens and Pseudomonas testosteroni have been determined to further delimit the origin of the endosymbiont that gave rise to the mitochondrion. These two prokaryotes represent the alpha and beta subdivisions, respectively, of the so-called purple bacteria. The endosymbiont that gave rise to the mitochondrion belonged to the alpha subdivision, a group that also contains the rhizobacteria, the agrobacteria, and the rickettsias--all prokaryotes that have developed intracellular or other close relationships with eukaryotic cells.
Domain structure and associated functions of subcomponents C1r and C1s of the first component of human complement.
Authors: Villiers C L , Arlaud G J , Colomb M G
Published: May 2006

The serine protease subcomponents of the activated form of the first component of human complement (C1), C1r and C1s, were observed by electron microscopy after the native proteins and their limited proteolysis products, obtained from autolytic cleavage (C1r) or from incubation with plasmin (C1s) were rotary shadowed. At the monomeric level, both C1r and C1s comprised two globular domains, a smaller interaction domain (corresponding to the NH2-terminal half of the A chain, alpha, and responsible for calcium binding and C1r-C1s interaction) and a larger catalytic domain (corresponding to the COOH-terminal part of the A chain, gamma, disulfide-linked to the B chain and bearing the serine protease active site). The two globular domains are linked by a connecting strand, beta. The (C1r)2 dimer appeared as a "croissant"-like association, where the two monomers interact through their catalytic domains. On the basis of the domain structure of C1r and C1s, a model of the calcium-dependent C1s dimer is proposed, in which the two monomers interact through their NH2-terminal interaction domains; in the same way, a model of the C1s-(C1r)2-C1s catalytic subunit of C1 is presented, in which (C1r)2 forms a core, its distal interaction domains interacting with the corresponding domains of C1s.
Epidermal growth factor stimulates glycogen synthase activity in cultured cells.
Authors: Chan C P , Krebs E G
Published: May 2006

Addition of epidermal growth factor (EGF) to quiescent cultured cells was found to stimulate the activity of glycogen synthase (UDPglucose:glycogen 4-alpha-D-glucosyltransferase, EC 2.4.1.11), an enzyme subjected to regulation by covalent modification. In Swiss mouse 3T3 cells, the activation by EGF paralleled the effect seen with insulin; the time course and dose-response curves of the two polypeptide factors were similar. Stimulation of enzyme activity ratio [(activity in the absence of glucose 6-phosphate)/(activity in the presence of glucose 6-phosphate)] was maximal after 20-30 min of incubation. Both factors caused a maximal stimulation of 2.5-fold in synthase activity ratio at approximately equal to 10 nM, and the half-maximal effect was observed at 0.1-1 nM. Insulin and EGF exhibited partial additivity in effecting this enzyme activation. In contrast, human A431 cells showed no response to insulin. Although quantitatively different, the EGF effect in the latter cells was time dependent, reaching a maximum at 90 min, and dose dependent, with a maximal stimulation of 4-fold in synthase activity ratio at 10 nM. Half-maximal effect was observed at 0.3 nM EGF. Direct quantitation of allosteric effectors (glucose 6-phosphate, adenine nucleotides, and Pi) present in the enzyme assay mixtures indicated that the observed activation was not simply a consequence of changes in metabolite concentrations. These results suggest that EGF may be important in regulating glycogen synthesis through phosphorylation/dephosphorylation mechanisms.
Sequence analysis of three Sindbis virus mutants temperature-sensitive in the capsid protein autoprotease.
Authors: Hahn C S , Strauss E G , Strauss J H
Published: May 2006

We have cloned and sequenced the cDNA made to the region of RNA encoding the structural proteins of three complementation group C mutants of Sindbis virus, ts2, ts5, and ts13, and of their revertants. These mutants possess defects in the posttranslational processing of their structural proteins at the nonpermissive temperature. Comparison of the deduced amino acid sequences of the mutants with those of the revertants and with the parental HR strain of virus showed all three mutants to have single amino acid substitutions in the highly conserved COOH-terminal half of the capsid protein that give rise to temperature sensitivity. ts2 and ts5 were found to have the same lesion and thus represent independent isolations of the same mutant, whereas ts13 possessed a different change. Reversion to temperature insensitivity in all three mutants occurred by reversion of the mutated nucleotide to the parental nucleotide, restoring the original amino acid. It has been previously postulated that the capsid protein possesses an autoproteolytic activity that cleaves the capsid protein from the nascent polyprotein during translation. Comparison of the amino acid sequence of the capsid protein with that of serine proteases leads us to hypothesize that histidine-141, aspartate-147, and serine-215 of the Sindbis capsid protein form the catalytic triad of a serine protease. This hypothesis is supported by the finding that all three temperature-sensitive lesions mapped occur near these residues: ts2 and ts5 change proline-218 to serine and in ts13 lysine-138 has been replaced by isoleucine.
Nucleosome core particles suppress the thermal untwisting of core DNA and adjacent linker DNA.
Authors: Morse R H , Cantor C R
Published: May 2006

Covalently closed circular DNA is known to undergo a temperature-dependent change in helical twist. We have reconstituted nucleosome core particles onto closed circular DNA and measured the thermal untwisting of the DNA as a function of nucleosome density. The results demonstrate that the DNA associated with the nucleosome core particle does not alter its twist when the temperature is varied between 4 degrees C and 37 degrees C, and that the length of DNA prevented from thermal untwisting includes the linker as well as the core DNA.
Synchrotron radiation x-ray scattering in the early stages of in vitro collagen fibril formation.
Authors: Suarez G , Oronsky A L , Bordas J , Koch M H
Published: May 2006

The time course of in vitro collagen fibril formation was monitored by synchrotron radiation x-ray scattering. Collagen polymerization was induced by a temperature jump from 4 degrees C to 32 degrees C and the solution scattering pattern was recorded continuously with a time resolution of a few seconds. The scattered intensity increased as soon as the final temperature was attained, without discernible lag phase, when the collagen concentration was about 0.56 mg/ml, whereas turbidimetric measurements revealed a lag phase of ca. 2.5 min at 1.05 mg/ml. A direct correlation was found between the temperature and both the rate of formation and the total amount of early aggregates. The formation of these aggregates was only partially reversed by lowering the temperature, except when the collagen had been prevented from forming Schiff base-mediated intermolecular crosslinks by sodium borohydride reduction. In this case, formation of aggregates was completely reversible. We conclude that the aggregates that appear to correspond to the subfibrils proposed on the basis of independent methods are formed and simultaneously crosslinked in the early phases of in vitro collagen self-assembly.