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1.  A Role for Short-Term Synaptic Facilitation and Depression in the Processing of Intensity Information in the Auditory Brain Stem 
Journal of Neurophysiology  2007;97(4):2863-2874.
The nature of the synaptic connection from the auditory nerve onto the cochlear nucleus neurons has a profound impact on how sound information is transmitted. Short-term synaptic plasticity, by dynamically modulating synaptic strength, filters information contained in the firing patterns. In the sound-localization circuits of the brain stem, the synapses of the timing pathway are characterized by strong short-term depression. We investigated the short-term synaptic plasticity of the inputs to the bird’s cochlear nucleus angularis (NA), which encodes intensity information, by using chick embryonic brain slices and trains of electrical stimulation. These excitatory inputs expressed a mixture of short-term facilitation and depression, unlike those in the timing nuclei that only depressed. Facilitation and depression at NA synapses were balanced such that postsynaptic response amplitude was often maintained throughout the train at high firing rates (>100 Hz). The steady-state input rate relationship of the balanced synapses linearly conveyed rate information and therefore transmits intensity information encoded as a rate code in the nerve. A quantitative model of synaptic transmission could account for the plasticity by including facilitation of release (with a time constant of ~40 ms), and a two-step recovery from depression (with one slow time constant of ~8 s, and one fast time constant of ~20 ms). A simulation using the model fit to NA synapses and auditory nerve spike trains from recordings in vivo confirmed that these synapses can convey intensity information contained in natural train inputs.
PMCID: PMC3268177  PMID: 17251365
2.  Filamentous phage replication initiator protein gpII forms a covalent complex with the 5' end of the nick it introduced. 
Nucleic Acids Research  1999;27(8):1882-1889.
Rolling circle type DNA replication is initiated by introduction of a nick in the leading strand of the origin by the initiator protein, which in most cases binds covalently to the 5' end of the nick. In filamentous phage, however, such a covalent complex has not been detected. Using a suitable substrate and short reaction time, we show that filamentous phage initiator gpII forms a covalent complex with nicked DNA, which rapidly dissociates unless gpII is inactivated. A peptide-DNA complex was isolated from trypsin digest of the complex by ion-exchange column chromatography and gel filtration, and its peptide sequence was determined. The result indicated that gpII was linked to DNA by the tyrosine residue at position 197 from the N-terminus. The mutant protein in which this tyrosine was replaced by phenylalanine did not show any detectable activity to complement gene II amber mutant phage in vivo. In vitro, the mutant protein recognized the origin and bent DNA as well as the wild-type does, but failed to introduce a nick and to relax the superhelicity of cognate DNA.
PMCID: PMC148397  PMID: 10101197
3.  A promoter for the first nine genes of the Escherichia coli mra cluster of cell division and cell envelope biosynthesis genes, including ftsI and ftsW. 
Journal of Bacteriology  1997;179(18):5802-5811.
We constructed a null allele of the ftsI gene encoding penicillin-binding protein 3 of Escherichia coli. It caused blockage of septation and loss of viability when expression of an extrachromosomal copy of ftsI was repressed, providing a final proof that ftsI is an essential cell division gene. In order to complement this null allele, the ftsI gene cloned on a single-copy mini-F plasmid required a region 1.9 kb upstream, which was found to contain a promoter sequence that could direct expression of a promoterless lacZ gene on a mini-F plasmid. This promoter sequence lies at the beginning of the mra cluster in the 2 min region of the E. coli chromosome, a cluster of 16 genes which, except for the first 2, are known to be involved in cell division and cell envelope biosynthesis. Disruption of this promoter, named the mra promoter, on the chromosome by inserting the lac promoter led to cell lysis in the absence of a lac inducer. The defect was complemented by a plasmid carrying a chromosomal fragment ranging from the mra promoter to ftsW, the fifth gene downstream of ftsI, but not by a plasmid lacking ftsW. Although several potential promoter sequences in this region of the mra cluster have been reported, we conclude that the promoter identified in this study is required for the first nine genes of the cluster to be fully expressed.
PMCID: PMC179470  PMID: 9294438
4.  Carcinoma of stomach and breast with lymphoid stroma: localisation of Epstein-Barr virus. 
Journal of Clinical Pathology  1994;47(6):538-540.
AIMS--To determine the presence of Epstein-Barr virus (EBV) genome in six patients (three with gastric and three with breast carcinoma) with severe small lymphoid cell infiltration. METHODS--The polymerase chain reaction and in situ hybridisation were used to detect EBV genome. The number and distribution of T and B lymphocytes were evaluated by immunohistochemistry. RESULTS--Histologically all of the patients had poorly differentiated tumours. Immunohistochemistry showed that T cells predominated in three cases, B cell in two, and almost equal numbers in one case. PCR showed that the EBV genome was present in two cases each of gastric and breast carcinoma. In situ hybridisation for EBV genome provided positive signals only in the small lymphoid cells in one gastric and two breast carcinomas giving a positive reaction for EBV genome by PCR. The gastric and breast cancer cells did not give positive signals. CONCLUSIONS--Severe lymphoid cell infiltration in gastric and breast carcinoma does not necessarily indicate that these tumours are associated with EBV. Larger numbers of cases will need to be studied to confirm this.
PMCID: PMC494746  PMID: 8063937
5.  SOS induction in Escherichia coli by single-stranded DNA of mutant filamentous phage: monitoring by cleavage of LexA repressor. 
Journal of Bacteriology  1995;177(12):3610-3612.
Infection of Escherichia coli in the presence of chloramphenicol with mutant filamentous phage that are defective in the initiation of minus-strand DNA synthesis induces the SOS response as monitored by cellular LexA levels. This observation demonstrates that single-stranded DNA serves as a primary signal for SOS induction in vivo.
PMCID: PMC177072  PMID: 7768876
6.  Nucleotide sequence of the primer RNA for DNA replication of filamentous bacteriophages. 
Journal of Virology  1993;67(4):2175-2181.
We determined the nucleotide sequence of RNA synthesized in vitro by Escherichia coli RNA polymerase at the complementary-strand replication origin on the single-stranded viral DNA of bacteriophages f1 and IKe (ori-RNA) by using chain-terminating ribonucleoside triphosphate analogs. The results indicated that the start site of f1 ori-RNA synthesis is 20 nucleotides downstream from the site previously reported (K. Geider, E. Beck, and H. Schaller, Proc. Natl. Acad. Sci. USA 75:645-649, 1978) and that the RNA sequence [(5')pppAGGGCGAUGGCCCACUACGU-OH(3')] is complementary to the f1 DNA sequence from nucleotides 5736 to 5717, with minor heterogeneity at the 3' end. IKe ori-RNA had a sequence identical to that of f1 ori-RNA, except for a single base substitution, and IKe RNA was complementary to a region of IKe DNA (from nucleotides 6441 to 6422) that was homologous to the f1 sequence. Phenotypes and ori-RNA sequences in the relevant region of the genome of f1 deletion mutants were consistent with the presently determined sequence of ori-RNA. A possibility that ori-RNA synthesis is initiated by a mechanism similar to that for general transcription is suggested as a result of the new assignment of the ori-RNA start site. The double-origin plasmid assay of minus-strand origin activity, a sensitive in vivo method for detecting cis-acting elements for the initiation of DNA replication on a single-stranded DNA template, is described.
PMCID: PMC240330  PMID: 8445727
7.  Correlation of a subset of the pLC plasmids to the physical map of Escherichia coli K-12. 
Microbiological Reviews  1992;56(1):137-151.
We determined map positions of the Escherichia coli K-12 portions of a subset of the hybrid E. coli-ColE1 plasmids constructed by Clarke and Carbon. The probe DNA of pLC plasmids was labeled with digoxigenine-dUTP, hybridized to the 476 phage clones of the E. coli ordered clone bank miniset, which was adsorbed on a strip of nylon membrane filters, and detected by enzyme-linked immunoassay and a subsequent enzyme-catalyzed color reaction. The total number of Clarke-Carbon plasmids we analyzed was 518, for which chromosomal locations of 297 clones were newly determined in the present study. Another 180 plasmids gave results that agreed with those reported previously, and the remaining 41 plasmids gave map positions different from those described in the previous report. A chromosome map of E. coli which shows the locations of 518 pLC plasmids on it is presented, as well as a table which correlates the pLC plasmids with the clones of the E. coli ordered clone bank miniset on the basis of the hybridization data. We estimate that approximately one-half of the entire genome of E. coli was covered by the pLC plasmids used in this study.
PMCID: PMC372858  PMID: 1579107
8.  SOS induction in Escherichia coli by infection with mutant filamentous phage that are defective in initiation of complementary-strand DNA synthesis. 
Journal of Bacteriology  1992;174(5):1612-1618.
We report that the SOS response is induced in Escherichia coli by infection with mutant filamentous phage that are defective in initiation of the complementary (minus)-strand synthesis. One such mutant, R377, which lacks the entire region of the minus-strand origin, failed to synthesize any detectable amount of primer RNA for minus-strand synthesis. In addition, the rate of conversion of parental single-stranded DNA of the mutant to the double-stranded replicative form in infected cells was extremely slow. Upon infection, R377 induced the SOS response in the cell, whereas the wild-type phage did not. The SOS induction was monitored by (i) induction of beta-galactosidase in a strain carrying a dinD::lacZ fusion and (ii) increased levels of RecA protein. In addition, cells infected with R377 formed filaments. Another deletion mutant of the minus-strand origin, M13 delta E101 (M. H. Kim, J. C. Hines, and D. S. Ray, Proc. Natl. Acad. Sci. USA 78:6784-6788, 1981), also induced the SOS response in E. coli. M13Gori101 (D. S. Ray, J. C. Hines, M. H. Kim, R. Imber, and N. Nomura, Gene 18:231-238, 1982), which is a derivative of M13 delta E101 carrying the primase-dependent minus-strand origin of phage G4, did not induce the SOS response. These observations indicate that single-stranded DNA by itself induces the SOS response in vivo.
PMCID: PMC206557  PMID: 1537803
9.  Immunochemical studies and complete amino acid sequence of the streptokinase from Streptococcus pyogenes (group A) M type 12 strain A374. 
Infection and Immunity  1992;60(1):278-283.
The complete amino acid sequence of the streptokinase (SKase) of Streptococcus pyogenes M type 12 strain A374, isolated from a patient with poststreptococcal glomerulonephritis (PSGN), was determined. The epitope domain for the monoclonal antibody N-59, which cross-reacts with SKases of both the PSGN-associated strain and S. equisimilis H46A (a non-PSGN-associated strain), was predicted to be localized in residues 370 to 374. The epitope domain specific for monoclonal antibody RU-1, which reacts only with the PSGN-associated SKase, was localized to residues 164 to 236.
PMCID: PMC257533  PMID: 1370275
10.  The mutH gene regulates the replication and methylation of the pMB1 origin. 
Journal of Bacteriology  1991;173(10):3209-3214.
DNA methylation is known to regulate several prokaryotic replication origins. In particular, the Escherichia coli chromosomal origin oriC and the pMB1 plasmid origin (which is homologous to the ColE1 origin) replicate poorly when hemimethylated at dam (GATC) sites. Because the mismatch repair protein MutH is known to recognize hemimethylated dam sites, its role in the replication of these origins was investigated. The results presented here show that the mutH gene product is partially responsible for the poor replication of the pMB1 origin when hemimethylated but has no effect on the replication of oriC. Methylation levels at individual dam sites suggest that the MutH protein binds to an inverted repeat in the pMB1 replication primer promoter. These findings suggest a mechanism for the coordinated control of DNA repair and replication.
PMCID: PMC207916  PMID: 2022619
12.  Endonuclease R-EcoRII restriction of bacteriophage f1 DNA in vitro: ordering of genes V and VII, location of an RNA promotor for gene VIII. 
Journal of Virology  1975;16(3):674-684.
Replicative form DNA of bacteriophage f1 was found to be sensitive in vitro to restriction by endonuclease R-EcoRII if the DNA was isolated from an Escherichia coli strain deficient in cytosine methylase activity. A similar observation was previously made with DNA from the closely related bacteriophage fd (S. Schlagman, S. Hattman, M. S. May, and L. Berger, submitted for publication). The two DNA fragments produced by the endo R-EcoRII digestion of f1 DNA were localized on the f1 cleavage map and their genetic content was determined. The polypeptides synthesized in a "coupled" transcription-translation system under the direction of each RII fragment were examined. The results of such experiments allow the ordering of genes V and VII and indicate the location of a RNA promotor for gene VIII.
PMCID: PMC354715  PMID: 1159896
14.  A single amino acid substitution reduces the superhelicity requirement of a replication initiator protein. 
Nucleic Acids Research  1992;20(11):2685-2691.
The origin of rolling circle replication in filamentous coliphage consists of a core origin that is absolutely required and an adjacent replication enhancer sequence that increases in vivo replication 30 to 100-fold. The core origin binds the initiator protein (gpII) which either nicks or relaxes negatively superhelical replicative form DNA (RFI). Nicking at the origin, but not relaxation, leads to initiation of DNA replication. Our results indicate that the ratio of nicking to relaxation (nicking-closing) in vitro depends on the superhelical density of the substrate. We have studied the effect of a single amino acid substitution in gpII, which allows wild-type levels of replication in the absence of the enhancer, on origin nicking and binding. The enhancer-independent mutation yields more nicking and less relaxation of RFI, compared to the wild-type protein. The mutant gpII also shows a reduced requirement for superhelicity of the substrate in the nicking reaction. At the same time, the mutant gpII increases the cooperativity of protein-protein interactions in origin binding. We propose that the relaxation activity of gpII negatively regulates replication initiation, and that both increase in the negative superhelicity of the substrate and action of the replication enhancer may antagonize the relaxation activity.
PMCID: PMC336908  PMID: 1614854

Results 1-14 (14)