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A computer method for construction of secondary structure from polynucleotide sequence. Possible structure of the bacterial replication origin.

A computer method to search the possible secondary structure of a long polynucleotide was developed. As a criterion for the stabilization of a secondary structure, free energy originating from base-pairing was employed, since the structure in solution would be at the free energy minimum. The method is summarized as follows: all possible helices are collected from a given nucleotide sequence under restrictions that the length of a helix is greater than N0 bases (e.g., four bases) and the free energy of the helix calculated according to free energies of two successive sequence-dependent basepairs is lower than E0 (e.g., -5 kcal/mol). The search of secondary structures of low free energy is performed by connecting one helix to another without allowing any base-pairing between loops. For connecting single-stranded regions, destabilizing free energy of 2--3 kcal/mol is added. The method was first applied to several tRNAs and the clover-leaf structure of tRNA was obtained as a free energy minimum. Then, possible secondary structures of the replication origin regions of the Escherichia coli and Salmonella typhimurium chromosomes were examined by the method, assuming that one of the strands in the origin region takes a specific secondary structure. The lowest-energy structure for the E. coli origin was found to be approximately identical to that for the S. typhimurium origin region.

Base Sequence↗

Laminar origins and terminations of cortical connections of the occipital lobe in the rhesus monkey.

Cortical connections within the occipital lobe (areas 17, 18 and 19) of the rhesus monkey are investigated with the autoradiographic and horseradish peroxidase procedures. Two efferent systems, each with a specific laminar organization, are observed. (1) Rostrally directed connections, from area 17 to 18, area 18 to 19 and area 19 to the inferotemporal region (area TE), originate from neurons in layer IIIc (and, in area 19, from a small complement of neurons in layer Va), and terminate in and around layer IV. (2) In contrast, connections in the reverse direction ('caudally directed' connections), from area TE to 19, area 19 to 18, and area 18 to 17, originate from neurons in layers Vb, VI and, to a lesser extent, IIIa, and terminate mainly in layer I. In addition, the laminar organization of several intrinsic and callosal connections are observed. In trinsic connections within areas 18 and 19 originate from neurons in layers IIIc and, to a lesser extent, Va, and terminate in vertical bands in layers I to IV. Callosal connections from areas 18, 19, and the caudal inferotemporal region originate from neurons mainly in layer IIIc. From areas 18 and 19, these callosal connections terminate in vertical bands in layers I through IV. Thus, different cortical projection systems are characterized by specific laminar distributions of efferent terminations as well as of their neurons of origin.

Animals↗

The cells of origin of the hypoglossal afferent nerves and central projections in the cat.

The cells of origin for the hypoglossal afferent nerves of the cat and their central projections were examined using the transganglionic and somatopetal transport of horseradish peroxidase (HRP). Primary afferent neurons from the hypoglossal nerve were located in the trigeminal ganglion, the superior ganglion of glossopharyngeal and vagal nerves, and the first 3 cervical ganglia. The central projections of hypoglossal afferents were organized in a selective manner according to their cells of origin. The primary afferent nerves originating from the trigeminal ganglion terminated in the subnucleus dorsalis (Vpd) of the principal nucleus (Vp), lateral margin of the caudal pars interpolaris (Vi), interstitial nucleus and laminae I and V of the pars caudalis (Vc). The projection of the afferent nerves for glossopharyngeal and vagal origins are similarly organized in the Vi and Vc to those of trigeminal origin, but differed in that they terminated ipsilaterally in the caudal half of the solitary nucleus and bilaterally in the commissural nucleus. The primary afferents arising from the first 3 cervical ganglia terminated in laminae I and V of the corresponding cervical cord segments.

Animals↗

Long-lasting changes in the origin of spontaneous discharges from amygdala-kindled rats: piriform vs. perirhinal cortex in vitro.

The origin of spontaneous field potentials in coronal slices of the amygdala-piriform-perirhinal area (A-P area) from amygdala-kindled and control rats was assessed. In Expt. 1, the field potentials initially originated in the perirhinal (PRh) cortex of control tissue while they originated in the piriform (Pir) cortex of amygdala-kindled tissue. In Expt. 2, this kindling-based change was observed in the A-P area ipsilateral but not contralateral to the kindled amygdala. In both experiments, subsequent exposure to perfusion medium containing 0 Mg2+ resulted in the genesis of strong discharges in both control and kindled tissues. After 2-3 h of such treatment, the origin of spontaneous discharges in control tissue changed from the PRh to the Pir area and persisted in a reduced form during reperfusion with medium containing Mg2+. This change in origin of the discharges in control tissue appeared similar to that seen in previously kindled tissue. In Expt. 3, during exposure to 0 Mg2+, the response of the basolateral amygdala (BLA) was compared with the Pir and PRh areas. Independent of the PRh discharge, the BLA discharge closely followed the Pir discharge both in time and morphology. These lasting changes in the ipsilateral A-P area in vitro must be related in vivo to the change which allows the kindled A-P area to participate in the triggering of generalized limbic-kindled convulsions.

Amygdala↗

Comparison of the performances of an automated arrhythmia detector working on original and virtual ECG tracings.

Two approaches can be taken to improve the performance of an automatic arrhythmia detector: perfecting the detection algorithms or improving the quality of the investigated traces by preprocessing the original traces. This paper reports on the results of a data preprocessing approach. Preprocessing consists in constructing new traces, which we call virtual. They are mathematically obtained from the original traces and referred to the dominant cardiac electric axis. The classifications obtained with an arrhythmia detector using both virtual and original traces are presented and discussed. By comparing the performance indices obtained under the two different conditions, it can be seen that a diagnosis based on the virtual traces is as acceptable as one based on the original traces. This result should be judged as favorable, since the algorithm was not adjusted or calibrated to the virtual traces, while those who developed it had certainly calibrated the parameters to the original traces.

Algorithms↗

The relationship between chromosomal origins of replication and the nuclear matrix during the cell cycle.

A cytological investigation into the dynamic behaviour of the origins of replication with respect to the nuclear matrix has been carried out on Xenopus laevis cultured cells. In order to preferentially label origins or 'non-origin' regions along DNA fibres, 5-fluoro-2'-deoxyuridine (FUdR)-treated cells were pulsed with [3H]deoxyadenosine in early or late S phase. Samples were then allowed to proceed through the cell cycle for increasing times. The DNA loops were induced in situ to completely uncoil around the nuclear matrix. The autoradiographic analysis shows that, under the experimental conditions used, 'non-origin' regions behave as expected from previous studies, i.e., they associate with the nuclear matrix only when they become part of a replication fork, whereas active origins of replication remain associated with the matrix throughout the cell cycle.

Animals↗

Geographic origins of Jewish patients with inflammatory bowel disease.

Ulcerative colitis and Crohn's disease are more common among Jews than among non-Jews. Despite the existence of studies on the prevalence and incidence of inflammatory bowel disease in relation to the continent of residence or origin, there are no studies on the specific countries of origin of Ashkenazi Jewish patients. We report here the first analysis of 233 U.S. Jewish patients by defined world regions and subregions. Using two different sets of controls (a self-referred Jewish population for Tay-Sachs disease carrier detection and a sample of Jewish persons ascertained through unrelated studies from the same hospital as our patients), we found a significant excess of patients of middle European origin relative to those of Polish or Russian origin. These observations suggest that the inflammatory bowel disease gene(s) are more prevalent in the Jewish population that originated in middle Europe than in those from Poland and Russia. These results further suggest that Jewish patients with inflammatory bowel disease probably represent a nonrandom genetically predisposed subset of the Jewish population. This provides further evidence for the genetic contribution to inflammatory bowel disease in general, and to its higher risk in the Jewish population.

Colitis, Ulcerative↗

Characterization of a defective phage system for the analysis of bacteriophage T4 DNA replication origins.

We have developed a defective phage system for the isolation and analysis of phage T4 replication origins based on the T4-mediated transduction of plasmid pBR322. During the initial infection of a plasmid-containing cell, recombinant plasmids with T4 DNA inserts are converted into fully modified linear DNA concatamers that are packaged into T4 phage particles, to create defective phage (transducing particles). In order to select T4 replication origins from genomic libraries of T4 sequences cloned into the plasmid pBR322, we searched for recombinant plasmids that transduce with an unusually high efficiency, reasoning that this should select for T4 sequences that function as origins on plasmid DNA after phage infection. We also selected for defective phage that can propagate efficiently with the aid of a coinfecting helper phage during subsequent rounds of phage infection, which should select for T4 sequences that can function as origins on the linear DNA present in the defective phage. Several T4 inserts were isolated repeatedly in one or both of these selective procedures, and these were mapped to particular locations on the T4 genome. When plasmids were selected in this way from genomic libraries constructed using different restriction nucleases, they contained overlapping segments of the T4 genome, indicating that the same T4 sequences were selected. The inserts in two of the selected plasmids permit a very high frequency of transduction from circular plasmids; these have been shown to contain a special type of T4 replication origin.

DNA Replication↗

Interaction between gene II protein and the DNA replication origin of bacteriophage f1.

The origin of DNA replication of the filamentous bacteriophage f1 binds its initiator protein (gene II protein) in vitro to form a complex that can be trapped on nitrocellulose filters. The binding occurs with both superhelical form DNA and linear DNA fragments. A number of defective mutants of the origin were tested for the ability to bind gene II protein. The region of DNA required for the binding is around a second palindrome downstream from the palindrome that contains the DNA replication initiation site. It overlaps, but is not identical to, the region required for the nicking reaction by the protein. The nicking site itself was dispensable for the binding. In vivo, a number of defective deletion mutants of the origin, when in a plasmid, inhibited growth of superinfecting phage if the intracellular level of gene II protein was low. In addition, these defective origins inhibited the activity of the functional phage origin located on the same replicon. The domain of the DNA sequence required for inhibition in vivo was consistent with that for the binding in vitro.

Bacteriophages↗

Interaction between the replication origin and the initiator protein of the filamentous phage f1. Binding occurs in two steps.

The replication initiator protein of bacteriophage f1 (gene II protein) binds to the phage origin and forms two complexes that are separable by polyacrylamide gel electrophoresis. Complex I is formed at low gene II protein concentrations, and shows protection from DNase I of about 25 base-pairs (from position +2 to +28 relative to the nicking site) at the center of the minimal origin sequence. Complex II is produced at higher concentrations of the protein, and has about 40 base-pairs (from -7 to +33) protected. On the basis of gel mobility, complex II appears to contain twice the amount of gene II protein as does complex I. The 40 base-pair sequence protected in complex II corresponds to the minimal origin sequence as determined by in-vivo analyses. The central 15 base-pair sequence (from +6 to +20) of the minimal origin consists of two repeats in inverted orientation. This sequence, when cloned into a plasmid, can form complex I, but not complex II. We call this 15 base-pair element the core binding sequence for gene II protein. Methylation interference with the formation of complex I by the wild-type origin indicates that gene II protein contacts six guanine residues located in a symmetric configuration within the core binding sequence. Formation of complex II requires, in addition to the core binding sequence, the adjacent ten base-pair sequence on the right containing a third homologous repeat. A methylation interference experiment performed on complex II indicates that gene II protein interacts homologously with the three repeats. In complex II, gene II protein protects from DNase I digestion not only ten base-pairs on the right but also ten base-pairs on the left of the sequence that is protected in complex I. Footprint analyses of various deletion mutants indicate that the left-most ten base-pairs are protected regardless of their sequence. The site of nicking by gene II protein is located within this region. A model is presented for the binding reaction involving both protein-DNA and protein-protein interactions.

Base Sequence↗

Mutational analysis of the bacteriophage phi X174 replication origin.

Bacteriophage phi X174 mutants within the 30 base-pair replication origin were constructed using oligodeoxynucleotide-directed mutagenesis. A total of 18 viable base substitution mutants at 13 different positions within the origin region were obtained. The majority of these ori mutants have a plaque morphology and burst size comparable to that of wild-type phi X174. Two phi X174 ori mutants with a reduced growth ability spontaneously acquired additional mutations that enhanced the growth rate. The additional mutation was located at the same site as the original mutation or was located in the N-terminal part of the gene A protein. This latter secondary mutation is responsible for a better binding and/or recognition of the gene A protein to the mutated origin. In a Darwinian experiment wild-type phi X174 outgrows all phi X174 ori mutants, indicating the superiority of the wild-type ori sequence for the reproduction of bacteriophage phi 174. Insertions and deletions were constructed at different positions within the phi X174 replication origin cloned in a plasmid. Small insertions and deletions in the A + T-rich spacer region do not inhibit phi X174 gene A protein cleavage in vitro, but severely impair packaging of single-stranded plasmid DNA in viral coats.

Bacteriophage phi X 174↗

Fasciculations and their F-response. Localisation of their axonal origin.

A fasciculation with an axonal origin may be followed by its own F-response. The time-lapse between these 2 potentials corresponds to the time for the antidromic wave to travel to the alpha-cell and to return to the trigger-point. This relatively stable interval may be compared with the difference between the latencies of the F- and M-responses of the same muscle evoked by distal nerve stimulation. This permits one to localise the origin of the fasciculation on the axon. This method showed that, of 201 fasciculations followed by their F-response, obtained in normal controls and in neurological patients, 97 and 89% respectively had their origin between the muscle and the stimulation site. In this series, the proportion of pathological fasciculations having an origin between the muscle and a point at half the M-latency, is similar to that found in a former study on unselected fasciculations using a collision method (Roth 1982). Comparison between these 2 studies therefore supports the concept that all or almost all fasciculations have an axonal origin.

Axons↗

Characterization of the TRS/IRS origin of DNA replication of herpes simplex virus type 1.

In previous experiments, an origin of viral DNA replication was localized within a 995-bp DNA fragment that mapped entirely within the TRS/IRS repeated region of the herpes simplex virus type 1 genome (N. D. Stow, EMBO J. 1, 863-867, 1982). In this paper, this origin is now shown to reside within a 535-bp segment subcloned from the above fragment. Deletions extending various distances into each end of the 535-bp segment were generated using nuclease BAL 31, and the resulting plasmids screened for their ability to replicate in cells superinfected with wild-type HSV-1 helper virus. This analysis indicated that the cis-acting sequences essential for DNA replication were present within a 90-bp region, and a 100-bp viral DNA fragment containing all the signals necessary for origin function was identified. The origin lies within an untranscribed region located between the 5'-ends of two divergently transcribed immediate-early mRNAs. A prominant feature of the origin region is an almost perfect palindromic sequence 45 bp long containing 18 consecutive A or T residues at its center.

Base Sequence↗

DNA looping induced by bacteriophage lambda O protein: implications for formation of higher order structures at the lambda origin of replication.

A plasmid has been constructed, pOri2, which contains two lambda replication origin sequences separated by 1068 bp; both lambda sequences having the same orientation. When lambda initiation protein O is reacted with linearized pOri2 and examined by electron microscopy it is found to contain a looped area in which two parts of the plasmid are bound together by the O protein complex. Length measurements show that the O protein binds at the expected positions of the lambda origin sequences and that the looped area represents the DNA segment between the two O protein binding domains. Similar looping occurs in reactions with supercoiled pOri2 or if an amino-terminal fragment of O protein is used. When looped molecules are reacted with psoralen, crosslinked by irradiation with uv light, and then denatured, it is found that the looped area is more thermostable than the rest of the molecule. This indicates that the DNA within the looped segment is torsionally constrained while that outside the loop is free to rotate and suggests that simultaneous binding of O to two origins fixes the linkage number of the intervening DNA. The double origin binding ability of O may be diagnostic of the details of the reaction of O with a single origin sequence. A model is presented that rests on the assumption that O can produce microscopic looping between O protein binding sites within a single ori sequence.

Bacteriophage lambda↗

Analysis of the binding sites for the varicella-zoster virus gene 51 product within the viral origin of DNA replication.

The C-terminal 322 amino acids of the varicella-zoster virus (VZV) gene 51 product were expressed in Escherichia coli and shown to bind to specific DNA sequences within the VZV origin of DNA replication. The gene 51 product and its herpes simplex virus type 1 homolog (the UL9 protein) are capable of recognizing identical DNA sequences but the arrangement of binding sites within the origin regions of the two viruses differs. Three binding sites for the VZV gene 51 protein were identified within the VZV origin region and these lie in the same orientation and on the same side of the origin palindromic DNA sequence. DNA replication assays in transfected tissue culture cells demonstrated that the site closest to the palindrome is essential for origin activity whereas the most distal site is dispensable. The middle binding site may play an auxiliary role in DNA synthesis.

Base Sequence↗

Intimal neovascularization in human coronary atherosclerosis: its origin and pathophysiological significance.

To investigate the histopathological characteristics of the newly formed vessels in the atherosclerotic intima of human coronary arteries, we conducted postmortem angiography in 31 cases, including 11 with myocardial infarction. Vessels were examined three-dimensionally under the stereoscope. In addition, we evaluated 25 anterior descending coronary arteries unrelated to the occurrence of myocardial infarction by light microscopy using 3-mm stepwise sections and 5-microns serial sections. Histological alterations were analyzed morphometrically to determine the correlation between the degree of intimal neovascularization and the growth of the endothelium into the atherosclerotic intima from the adventitia or lumen. There was a significant positive correlation between the density of new vessels in the intima and the incidence of luminal stenosis, the extent of chronic inflammatory infiltrate, the formation of granulation tissue, or the atheromatous changes, whereas the vascular density decreased in the extensively hyalinized and calcified intima. The newly formed intimal vessels originated mainly from the adventitial vasa vasorum and also partly from the proper coronary lumen. The intimal vessels that originated from the adventitia occurred approximately 28 times more frequently than those that originated from the luminal side. The frequency of former vessels increased as the luminal stenosis became more severe, whereas the latter vessels were found most frequently in the presence of 40% and 50% stenosis. Vessels originating from the proper lumen were more often associated with fresh or old hemorrhage. We conclude that intimal neovascularization largely originates from the adventitia and is closely associated with the extent of coronary stenosis and the histological inflammatory reaction.

Adult↗

Amplification and excision of integrated polyoma DNA sequences require a functional origin of replication.

Cells transformed by Polyoma virus (Py) can undergo a high rate of excision or amplification of integrated viral DNA sequences, and these phenomena require the presence of homology (i.e., repeats) within the viral insertion as well as a functional viral large T antigen (T-Ag). To determine whether the main role of large T-Ag in excision and amplification was replicative or recombination-promoting, we studied transformed rat cell lines containing tandem insertions of a ts-a Py molecule (encoding a thermolabile large T-Ag) with a deletion of the origin of viral DNA replication. Culturing of these cells at the temperature permissive for large T-Ag function did not result in any detectable excision or amplification of integrated Py sequences. We then introduced into origin-defective lines a recombinant plasmid containing the viral origin of replication and the gene coding for resistance to the antibiotic G418. All G418-resistant clones analyzed readily amplified the integrated plasmid molecules when grown under conditions permissive for large T-Ag function, showing that these cells produced viral large T-Ag capable of promoting amplification in trans of DNA sequences containing the Py origin. These observations strongly suggest that Polyoma large T antigen promotes excision or amplification of viral DNA by initiating replication at the integrated origin, providing a favorable substrate for subsequent recombination.

Animals↗

Regulation of regional cerebral blood flow by cholinergic fibers originating in the basal forebrain.

We review mainly recent studies on vasodilative regulation of cortex and hippocampus by central cholinergic nerves originating in the basal forebrain. We also briefly review the influence of other central noradrenergic fibers originating in the locus ceruleus, serotonergic fibers originating in the dorsal raphe nucleus, dopaminergic fibers originating in the substantia nigra, and peripheral sympathetic and parasympathetic nerve fibers upon regulation of regional cerebral blood flow. Local metabolites have long been considered to play an important physiological role in regulating regional cerebral blood flow. However, the evidence reviewed here emphasizes that the regulation of regional cerebral blood flow by these central cholinergic nerves is independent of regional metabolism. We propose through this review that although studies investigating neural regulation of cortical and hippocampal blood flow by cholinergic fibers originating in the basal forebrain have added much to the understanding of regulation of regional cerebral blood flow further studies are needed to determine the physiological relevance of regional cerebral blood flow in relation to higher nervous functions such as memory, learning, and personality, and changes in these cognitive functions with aging and pathology such as Alzheimer's disease.

Animals↗