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T Haug

Publications and source records attributed to T Haug.

At least 37 records · Page 2Linked to original sources
PubMed1997

A sequence in the N-terminal region of human uracil-DNA glycosylase with homology to XPA interacts with the C-terminal part of the 34-kDa subunit of replication protein A.

Uracil-DNA glycosylase releases free uracil from DNA and initiates base excision repair for removal of this potentially mutagenic DNA lesion. Using the yeast two-hybrid system, human uracil-DNA glycosylase encoded by the UNG gene (UNG) was found to interact with the C-terminal part of the 34-kDa subunit of replication protein A (RPA2). No interaction with RPA4 (a homolog of RPA2), RPA1, or RPA3 was observed. A sandwich enzyme-linked immunosorbent assay with trimeric RPA and the two-hybrid system both demonstrated that the interaction depends on a region in UNG localized between amino acids 28 and 79 in the open reading frame. In this part of UNG a 23-amino acid sequence has a significant homology to the RPA2-binding region of XPA, a protein involved in damage recognition in nucleotide excision repair. Trimeric RPA did not enhance the activity of UNG in vitro on single- or double-stranded DNA. A part of the N-terminal region of UNG corresponding in size to the complete presequence was efficiently removed by proteinase K, leaving the proteinase K-resistant compact catalytic domain intact and fully active. These results indicate that the N-terminal part constitutes a separate structural domain required for RPA binding and suggest a possible function for RPA in base excision repair.

Amino Acid Sequence↗
PubMed1997

Nuclear and mitochondrial uracil-DNA glycosylases are generated by alternative splicing and transcription from different positions in the UNG gene.

A distinct nuclear form of human uracil-DNA glycosylase [UNG2, open reading frame (ORF) 313 amino acid residues] from the UNG gene has been identified. UNG2 differs from the previously known form (UNG1, ORF 304 amino acid residues) in the 44 amino acids of the N-terminal sequence, which is not necessary for catalytic activity. The rest of the sequence and the catalytic domain, altogether 269 amino acids, are identical. The alternative N-terminal sequence in UNG2 arises by splicing of a previously unrecognized exon (exon 1A) into a consensus splice site after codon 35 in exon 1B (previously designated exon 1). The UNG1 sequence starts at codon 1 in exon 1B and thus has 35 amino acids not present in UNG2. Coupled transcription/translation in rabbit reticulocyte lysates demonstrated that both proteins are catalytically active. Similar forms of UNG1 and UNG2 are expressed in mouse which has an identical organization of the homologous gene. Constructs that express fusion products of UNG1 or UNG2 and green fluorescent protein (EGFP) were used to study the significance of the N-terminal sequences in UNG1 and UNG2 for subcellular targeting. After transient transfection of HeLa cells, the pUNG1-EGFP-N1 product colocalizes with mitochondria, whereas the pUNG2-EGFP-N1 product is targeted exclusively to nuclei.

Alternative Splicing↗
PubMed1997

BK channels in intact clonal rat pituitary cells are activated by physiological elevations of the cytosolic Ca2+ concentration at the normal resting potential.

Activation of large conductance Ca(2+)-activated K+ channels (BK channels) in intact clonal rat pituitary cells (GH4 cells) was investigated using the cell-attached patch-clamp configuration. This method prevents loss of intracellular factors which might influence channel activity. BK channels are generally considered to be inactive at the resting membrane potential in excitable cells. However, at the resting potential (0 mV pipette potential), 40% of the cell-attached patches displayed spontaneously active BK channels, which remained active even at 20 mV hyperpolarization. The peptide thyroliberin (TRH) elevates the cytosolic Ca2+ concentration ([Ca2+]i) in GH cells by IP3-induced release of Ca2+ from intracellular stores. This rise in [Ca2+]i occurs concomitantly with membrane hyperpolarization. TRH stimulation caused activation of BK channels in nine out of 30 silent cell-attached patches, and caused enhanced channel activity in seven out of 29 cell-attached patches containing spontaneously active BK channels. The Ca2+ ionophore ionomycin activated silent BK channels in three out of 10 cell-attached patches, and increased the activity of spontaneously active BK channels in seven out of 16 cell-attached patches. The pipette potential was clamped to 0 mV in all these experiments. We conclude that the BK channels in GH4 cells may be active at the resting membrane potential and more negative membrane potentials. The channels may also be activated further by physiological elevations of [Ca2+]i in the same potential range. Our results point towards new possible physiological roles for the BK channels in GH4 cells. This is in agreement with the emerging picture of BK channels highly sensitive to [Ca2+]i in a wide variety of cell types.

Animals↗
PubMed1996

Human uracil-DNA glycosylase gene: sequence organization, methylation pattern, and mapping to chromosome 12q23-q24.1.

The human uracil-DNA glycosylase gene (UNG) spans approximately 13.5 kb including the promoter. UNG comprises 6 exons and 5 introns and was assigned to chromosome 12q23-q24.1 by radiation hybrid mapping. UNG exhibits typical features of housekeeping genes, including a 5' CpG island of 1.2 kb and a very GC-rich TATA-less promoter containing a number of elements involved in constitutive expression and cell cycle regulation. A smaller CpG island is located just downstream of the gene. Within the 15-kb sequence we identified 16 Alu retroposons, 2 of which contain putative competent RNA polymerase III promoters, 3 copies of medium reiteration frequency repeats, and 1 copy of a mammalian-wide interspersed repetitive element, as well as a 300-bp TA-dinucleotide repeat. In vitro methylation of the UNG promoter strongly reduced promoter activity, but methylation may not be involved in regulation of UNG in vivo since a narrow region of the 5' CpG island comprising the putative transcription factor binding region appears to be invariably methylation-free.

Amino Acid Sequence↗
PubMed1996

Pseudogenes for the human uracil-DNA glycosylase on chromosomes 14 and 16.

Two clones containing nonfunctional pseudogenes for the human uracil-DNA glycosylase gene have been isolated. The sequences of the two clones that are homologous to the UNG cDNA span 670 and 580 bp, respectively. In the longest of these, a full length Sx type Alu sequence interrupts the homologous sequence. Chromosomal mapping locates the clones to chromosomes 16 and 14. Comparison of the pseudogene sequences to the cDNA sequence indicates that the pseudogenes diverged from the functional gene approximately 31 and 22 million years ago, which is before the point in evolution when great apes and hominides separated.

Base Sequence↗
PubMed1995

Properties of a recombinant human uracil-DNA glycosylase from the UNG gene and evidence that UNG encodes the major uracil-DNA glycosylase.

We have expressed a human recombinant uracil-DNA glycosylase (UNG delta 84) closely resembling the mature form of the human enzyme (UNG, from the UNG gene) in Escherichia coli and purified the protein to apparent homogeneity. This form, which lacks the first seven nonconserved amino acids at the amino terminus, has properties similar to a 50% homogeneous UDG purified from human placenta except for a lower salt optimum and a slightly lower specific activity. The recombinant enzyme removed U from ssDNA approximately 3-fold more rapidly than from dsDNA. In the presence of 10 mM NaCl, Km values were 0.45 and 1.6 microM with ssDNA and dsDNA, respectively, but Km values increased significantly with higher NaCl concentrations. The pH optimum for UNG delta 84 was 7.7-8.0; the activation energy, 50.6 kJ/mol; and the pI between 10.4 and 10.8. The enzyme displays a striking sequence specificity in removal of U from UA base pairs in M13 dsDNA. The sequence specificity for removal of U from UG mismatches (simulating the situation after deamination of C) was essentially similar to removal from UA matches when examined in oligonucleotides. However, removal of U from UG mismatches was in general slightly faster, and in some cases significantly faster, than removal from UA base pairs. Immunofluorescence studies using polyclonal antibodies against UNG delta 84 demonstrated that the major fraction of UNG was located in the nucleus. Furthermore, > 98% of the total uracil-DNA glycosylase activity from HeLa cell extracts was inhibited by the antibodies, indicating that the UNG protein represents the major uracil-DNA glycosylase in the cells.

Base Sequence↗
PubMed1994

Structure of the gene for human uracil-DNA glycosylase and analysis of the promoter function.

The gene for human uracil-DNA glycosylase (UNG) contains 4 exons and has an approximate size of 13 kb. The promoter is very GC rich and lacks a TATA box. Nested deletions of the promoter demonstrated that two SP1 elements and a putative c-MYC element proximal to the transcription initiation region were sufficient to support some 27% of the promoter activity, while a clone that in addition contained the elements E2F/SP1/CCAAT increased expression to almost 90% of the full-length construct. A region upstream of these elements appears to exert a negative control function.

Base Sequence↗
PubMed1991

Aversive and compensatory classical conditioning with diazepam as conditional stimulus.

The purpose of the first experiment was to investigate whether diazepam could acquire anxiogenic properties by signalling an aversive event. Rats were trained in an operant chamber in the pentylenetetrazol (PTZ) model of anxiety. Thereafter the animals were divided into groups that received classical diazepam conditioning (Group 1), and conditioning of diazepam + tone (Group 2). In the test phase diazepam was injected prior to placement in the operant chamber. Group 2 selected the PTZ-appropriate lever more often than the other groups, indicating that the tone induced anxiety, and diazepam did not. Tones and shock may therefore be more easily associated than diazepam and shock. The second experiment investigated this. Rats were trained the same way as in the first experiment. Thereafter the experimental group received injections of a small dose of diazepam prior to a second injection of a large dose of diazepam. The hypothesis was that a compensatory anxiogenic conditional response to diazepam's anxiolytic effect should be elicited by the small dose. There were no differences between the groups in lever selection, indicating that a compensatory anxiogenic response was not elicited.

Animals↗
PubMed1984

A cryofixation study of presumptive hygroreceptors on the antennule of a terrestrial isopod.

The structure of the apical sensilla on the antennule of the terrestrial isopod Porcellio scaber was examined in cryofixed and freeze substituted (CRF) and chemically fixed and dehydrated (CHF) material. CRF specimens generally showed a preservation superior to CHF material. Only in deeper regions did the tissue show damage from freezing. Each of the 13-22 sensilla contains two sensory cells. In contrast to earlier reports, it was observed that the dendritic segments of these cells are arranged in a unique, concentric manner. In CRF specimens the dendrites reach the tip of the sensilla and border upon the innermost layer of the complicated wall of the peg which is not pierced by pores. Silver-protein and lanthanum failed to penetrate the wall of the sensilla and also did not reach the dendrites via an apical pore, which therefore is regarded as a molting pore. The lymph spaces which, in CHF specimens, are observed around the dendrites and beneath the cuticle within the antennal tip are regarded as artefactual. From similarities in the dendritic structures to insect hygroreceptors and their relationship to the adjoining cuticle it can be assumed that the antennular sensilla in Porcellio are sensitive to humidity changes. Mechanoreception and chemoreception, however, cannot entirely be excluded as possible functions.

Animals↗
PubMed1984

Tolerance to the depressant effects of diazepam in the drug discrimination paradigm.

Seven groups of rats (n = 35) were run in operant drug experiments. All groups were trained on a Fixed Ratio 10 schedule to discriminate diazepam from saline. Two groups (n = 7, n = 6), after extensive drug discrimination training (doses of 2.0 and 3.0 mg/kg diazepam), were submitted to generalization experiments with various doses of the training drug. Two additional groups, (n = 6, n = 8) in the initial phase of drug discrimination, were trained on intermediate and high doses of diazepam (i.e., 5.0 and 10.0 mg/kg). The development of tolerance to the depressant effects of diazepam for these two groups was compared to the low dose sophisticated rats. Of the above-mentioned groups, two groups were given tests after a waiting period in drug discrimination training. In this test the two groups were compared to an additional group (n = 8) in its initial phases of drug discrimination training. The results show that a large number of low doses (i.e., doses below 3.0 mg/kg) is not able to induce any tolerance to the depressant effects of diazepam in this particular paradigm. Intermediate doses of diazepam (i.e., 3.0 mg/kg), administered in a large number, induced some tolerance to the depressant effects, while another intermediate dose (5.0 mg/kg) and a high dose (10.0 mg/kg) rapidly induced a significant tolerance. Once developed, the tolerance persisted for 51 days.

Analysis of Variance↗
PubMed1983

Neuropharmacological specificity of the diazepam stimulus complex: effects of agonists and antagonists.

A food-reinforced two-lever operant method was used to train rats (n = 10) to discriminate diazepam (3.0 mg/kg i.p.) from saline. Thereafter drugs with supposed agonist and antagonist activity were submitted for test. Pentobarbital was the only agonist able to produce the diazepam discriminative stimulus complex (DSC), while pentylenetetrazol was the only antagonist able to abolish the diazepam DSC. It is concluded that the diazepam DSC is highly specific in this test and is suitable for the investigation of the neuropharmacological action of the benzodiazepines.

Animals↗
PubMed1982

The diazepam stimulus complex: specificity in a rat model.

Rats were trained to discriminate between one dose of diazepam (2.0 mg/kg) and saline in a two-lever operant chamber. When the group (n = 7) reached the training criterion, other drugs were introduced to test. Test drugs with supposed agonistic effects included drugs sharing one or more effects with diazepam, while drugs with supposed antagonistic effects were selected in relation to both reported effects and a supposed neurochemical substrate for the diazepam discriminative stimulus complex (DSC). It is concluded that a discriminative stimulus complex induced by a low dose of diazepam is highly specific and dependent on specific elements in the training procedure as well as in the testing procedure.

Animals↗
PubMed1982

Onset and offset of the diazepam stimulus complex.

Rats were trained to discriminate 3.0 mg/kg diazepam from saline in a two lever operant procedure. The time from injection to test session was 30 minutes. The diazepam discrimination consisted of initial responses on the lever paired with saline, but after training shifted to the lever paired with diazepam (onset). When tested with saline immediately after injection, animals responded consistently on the saline lever throughout the test. A shift from the drug lever to the saline lever at a later time point was also observed (offset). In addition, it was not possible to establish a peripheral diazepam drug stimulus complex. The results show that diazepam exerts discriminative control from 10 to 210 minutes after intraperitoneal injections, confirming a central action of the diazepam drug stimulus complex. The method might be useful in experimentation on drug control of lever selection.

Animals↗
PubMed1981

Two opposite effects of diazepam on fear by differential training in the CER-paradigm.

Two groups of rats were trained in a CER paradigm. The conditioned stimulus was a sound, the unconditioned stimulus was an electric shock. Group 1 received conventional CER training before the effects of different doses of diazepam were studied. For Group 2 the shock was always and exclusively given contingent on pretreatment with diazepam. After prolonged training the compound thus became a discriminative stimulus complex (DSC) and produced response suppression during the CS. Group 2 was also challenged with various doses of diazepam. The results showed that diazepam acquired diametrically different properties in the two groups. Group 1 exhibited disinhibitory effects and Group 2 suppressive effects, which may reflect anxiolytic and anxiogenic properties, respectively.

Animals↗