Biomedical subjects

M Telkov

Publications and source records attributed to M Telkov.

4 recordsLinked to original sources

PubMed1998

Human prodynorphin gene generates several tissue-specific transcripts.

The present study analyzes the transcription of the human prodynorphin gene. Transfection experiments indicate that promoter activity for the 2.8 kb 'brain-type' human prodynorphin mRNA resides in the DNA region located 140-180 b upstream of the exon 1/intron A boundary and not 1.2 kb further upstream, as proposed by others [S. Horikawa, T. Takai, M. Toyosato, H. Takahashi, M. Noda, H. Kakidani, T. Kubo, T. Hirose, S. Tanayama, H. Hayashida, T. Miyata, S. Numa, Isolation and structural organization of the human preproenkephalin B gene, Nature 306, 1983, pp. 611-614]. The new data locates the human prodynorphin gene promoter for the brain-type mRNA in a position corresponding to the position of the rat prodynorphin gene promoter [J. Douglass, C.T. McMurray, J.E. Garett, J.P. Adelman, L. Calavetta, Characterization of the rat prodynorphin gene, Mol. Endocrinol. 3, 1989, pp. 2070-2078]. Three previously not described types of human prodynorphin mRNA of the same size, 2.8 kb, one expressed in fetal brain and two others in testis, were characterized in this study. These mRNAs are generated by alternative splicing of novel 5'-uppermost exons and transcription is probably initiated from other promoters. Human neuroblastoma SH-SY5Y and SK-N-MC cell lines previously used in studies of gene transcription have the 2.8 kb prodynorphin mRNA of adult brain alongside a more abundant, shorter 2.3 kb transcript. The latter transcript was also found in testis and in fetal brain. It lacked the 5'-part of the 2.8 kb mRNA including the signal peptide encoding sequence. The complex pattern of prodynorphin gene expression and its functional consequences are issues for further studies.

Base Sequence↗

PubMed1998

Preprodynorphin mRNA-expressing neurones in the rat parabrachial nucleus: subnuclear localization, hypothalamic projections and colocalization with noxious-evoked fos-like immunoreactivity.

The dorsal lateral subnucleus of the rat pontine parabrachial nucleus is a major target for ascending nociceptive information from the spinal cord. With in situ hybridization histochemistry, using a radiolabelled cRNA probe, we demonstrate that neurones in and near the dorsal lateral subnucleus express preprodynorphin mRNA. The cRNA probe was constructed from a PCR product amplified from rat genomic DNA. Sequencing of the PCR product revealed that it corresponded to the sequence 466-1101 of the rat preprodynorphin gene exon 4. Tract tracing experiments, using injection of cholera toxin subunit B into the hypothalamic median preoptic nucleus, showed a retrograde labelling pattern of neurones in the parabrachial nucleus that was almost identical to that of the preprodynorphin mRNA expressing neurones. Double-labelling, combining immunohistochemical detection of tracer and in situ hybridization, revealed that the retrogradely labelled neurones expressed preprodynorphin mRNA. A similar double-labelling, combining in situ hybridization with immunohistochemical detection of noxious-evoked fos following formalin injection into one hindpaw of awake animals, showed that almost all fos-immunoreactive neurones in the dorsal lateral parabrachial subnucleus also expressed preprodynorphin mRNA. Quantitative analysis suggested that the evoked fos immunoreactivity was accompanied by an increased preprodynorphin mRNA expression. The findings provide evidence that neurones in the dorsal lateral subnucleus produce dynorphin and project to the median preoptic nucleus, and that noxious stimulation in awake animals synaptically activates the dynorphinergic neurones in this subnucleus. These observations are consistent with the idea of a functional and chemical heterogeneity among different parabrachial subnuclei that serves to produce specific homeostatic responses to stimuli that changes the physiological status of the organism, including tissue damage.

Animals↗

PubMed1995

Characterization of human prodynorphin gene transcripts.

Prodynorphin gene transcripts have been characterized in human tissues with cRNA probes covering parts of the non-coding exon 1 and the main coding exon 4 using Northern blot and RNase protection experiments. A 2.8-kb signal was observed in human brain RNA with both the exon 1 and exon 4 probes. An RNase protection assay with the exon 1 probe, performed to map the 5' end of this transcript, produced protected fragments in the range of 0.11 to 0.15 kb indicating that exon 1 is 1.2 kb shorter than previously proposed. 5'-RACE-PCR and sequencing of amplified cDNA fragments confirmed this assignment. In adrenal gland, testis and the human small cell lung carcinoma cell line, U1690, several prodynorphin-like mRNAs structurally different from the brain prodynorphin mRNA species were observed.

Adrenal Glands↗

PubMed1995

[Leu5]enkephalin-encoding sequences are targets for a specific DNA-binding factor.

A DNA-binding factor with high affinity and specificity for the [Leu5]enkephalin-encoding sequences in the prodynorphin and proenkephalin genes has been characterized. The factor has the highest affinity for the [Leu5]-enkephalin-encoding sequence in the dynorphin B-encoding region of the prodynorphin gene, has relatively high affinity for other [Leu5]enkephalin-encoding sequences in the prodynorphin and proenkephalin genes, but has no apparent affinity for similar DNA sequences coding for [Met5]-enkephalin in the prodynorphin or proopiomelanocortin genes. The factor has been named [Leu5]enkephalin-encoding sequence DNA-binding factor (LEF). LEF has a nuclear localization and is composed of three subunits of about 60, 70, and 95 kDa, respectively. The highest levels were observed in rat testis, cerebellum, and spleen and were generally higher in late embryonal compared to newborn or adult animals. LEF activity was also recorded in human clonal tumor cell lines. LEF inhibited the transcription of reporter genes in artificial gene constructs where a [Leu5]enkephalin-encoding DNA fragment had been inserted between the transcription initiation site and the coding region of the reporter genes. These observations suggest that the [Leu5]enkephalin-encoding sequences in the prodynorphin and proenkephalin genes also have regulatory functions realized through interaction with a specific DNA-binding factor.

Aging↗