Biomedical subjects

T O Nielsen

Publications and source records attributed to T O Nielsen.

6 recordsLinked to original sources

PubMed2000

Circular YAC vectors containing short mammalian origin sequences are maintained under selection as HeLa episomes.

pYACneo, a 15.8-kb plasmid, contains a bacterial origin, G418-resistance gene, and yeast ARS, CEN, and TEL elements. Three mammalian origins have been cloned into this circular vector: 343, a 448-bp chromosomal origin from a transcribed region of human chromosome 6q; X24, a 4.3-kb element containing the hamster DHFR origin of bidirectional replication (oribeta), and S3, a 1.1-kb human anti-cruciform purified autonomously replicating sequence. The resulting constructs have been transfected into HeLa cells, and G418-resistant subcultures were isolated. The frequency of G418-resistant transformation was 1.7-8.7 times higher with origin-containing YACneo than with vector alone. After >45 generations under G418 selection, the presence of episomal versus integrated constructs was assessed by fluctuation assay and by PCR of supercoiled, circular, and linear genomic cellular DNAs separated on ethidium bromide-cesium chloride gradients. In stable G418-resistant subcultures transfected with vector alone or with linearized constructs, as well as in some subcultures transfected with circular origin-containing constructs, resistance was conferred by integration into the host genome. However, several examples were found of G418-resistant transfectants maintaining the Y.343 and the YAC.S3 circular constructs in a strictly episomal state after long-term culture in selective medium, with 80-90% stability per cell division. The episomes were found to replicate semiconservatively in a bromodeoxyuridine pulse-labeling assay for </=130 cell generations after transfection. Furthermore, after </=172 cell generations rescued episomal DNA could be isolated intact and unrearranged, and could be used to retransform bacteria. These versatile constructs, containing mammalian origins, have the capacity for further modification with human telomere or large putative centromere elements, in an effort to move towards construction of a human artificial chromosome.

Bromodeoxyuridine↗

PubMed2000

Paraganglioma of the tongue.

Paragangliomas are neuroendocrine tumors arising from extra-adrenal autonomic ganglia. We present what is to our knowledge the first immunohistochemically documented case of a paraganglioma of the tongue, a 2.5-cm benign tumor growing in the position of the foramen cecum. The patient was an elderly woman who presented with throat irritation. The histologic profile and ultrastructural appearance of this lesion were classic for paraganglioma, and cells stained strongly for neurosecretory granules by immunohistochemistry. Carcinoid, ectopic thyroid neoplasm, and other tumors that mimic paraganglioma were ruled out on immunohistochemical grounds. To our knowledge, paraganglia have not been previously documented in the tongue; this tumor may have arisen from a branch of cranial nerve VII or IX, or from an embryologic remnant of the thyroid or thyroid capsule.

Aged↗

PubMed1998

Guidelines for legalized euthanasia in Canada: a proposal.

Arguments for liberty, mercy, and dignity support the legalization of euthanasia, but there remains a possibility of undesirable social consequences should this occur. Accordingly, proposals must prevent involuntary euthanasia, prevent unconscious coercion of the terminally ill to request euthanasia, protect and enshrine the availability of first-class palliative care, ensure documentation for purposes of enforcement and study, and spell out enforceable consequences for violations. Guidelines set by the Royal Dutch Medical Association have largely failed to meet these requirements. In North America, proposals for legalization, such as Oregon's Measure 16 and the minority opinion in Canada's Rodriguez case, also have flaws in meeting these criteria. Legislation in the Northern Territory of Australia came closest to meeting the requirements outlined, but was overruled after a brief period in effect. In Canada, a comprehensive survey of current euthanasia practices and improved availability of palliative care must precede attempts at legalization. A specific proposal is made for ethics committees operating at a regional health board level to approve legal euthanasia fitting within careful guidelines. Composition, procedures and mandate are described. If a set of guidelines, balancing any right there is "to die with dignity" with a responsibility to protect the weakest in society, is proposed first by the medical community, Parliament may have the courage to enact legislation.

Australia↗

PubMed1997

Circular YAC vectors containing a small mammalian origin sequence can associate with the nuclear matrix.

Three different mammalian origins of DNA replication, 343, S3, and X24, have been cloned into a 15.8 kb circular yeast vector pYACneo. Subsequent transfection into HeLa cells resulted in the isolation of several stably maintained clones. Two cell lines, C343e2 and CS3e1, were found to have sequences maintained as episomes in long-term culture with a stability per generation of approximately 80%. Both episomes also contain matrix attachment region (MAR) sequences which mediate the binding of DNA to the nuclear skeleton and are thought to play a role in DNA replication. Using high salt extraction of the nucleus and fluorescent in situ hybridization, we were able to demonstrate an association of the 343 episome with the nuclear matrix, most probably through functional MAR sequences that allow an association with the nuclear matrix and associated regions containing essential replication proteins. The presence of functional MARs in small episomal sequences may facilitate the replication and maintenance of transfected DNA as an episome and improve their utility as small episomal constructs, potential microchromosomes.

Animals↗

PubMed1996

Identification of a putative DNA replication origin in the gamma-aminobutyric acid receptor subunit beta3 and alpha5 gene cluster on human chromosome 15q11-q13, a region associated with parental imprinting and allele-specific replication timing.

The region containing the GABAA receptor beta3 and alpha5 subunit-encoding genes is subject to parental imprinting and is organized in different allele-specific replication timing domains. A 60-kb domain displaying a maternal early/paternal late pattern of allele-specific replication timing asynchrony is nested within a larger region displaying the opposite pattern. The proximal portion of this maternal early replicating domain is incorporated into phage clone lambda84. In order to identify DNA structures which may be associated with the boundary between the replication domains, phage lambda84 has been subcloned into smaller fragments and several of these have been analyzed by nucleotide sequencing. A plot of helical stability for 13kb of contiguous sequence reveals several A + T-rich regions which display potential DNA unwinding. The plasmid subclones from phage lambda84 have been analyzed for bent DNA and one of these, p82, contains bent DNA and overlaps with the region of highest potential helical instability. Of the seven plasmids tested, only p82 shows strong autonomous replication activity in an in vitro replication assay, with replication initiating within the genomic insert. These results suggest that a putative origin of DNA replication contained within p82 may play a role in establishing the allele-specific replication timing domains in the GABAA receptor subunit gene cluster.

Alleles↗

PubMed1994

Autonomous replication in vivo and in vitro of clones spanning the region of the DHFR origin of bidirectional replication (ori beta).

Plasmids containing the origin of bidirectional replication (ori beta) of the Chinese hamster dihydrofolate reductase-encoding gene (DHFR) were tested for autonomous replication in vivo and in vitro. The results show that plasmids pX24 and pneoS13, that contain a 4.8- and a 11.5-kb fragment, respectively, spanning the ori beta region, are able to replicate autonomously in human cells and in a cell-free system that uses human cell extracts. Another plasmid, pX14, containing a 4.8-kb fragment that is immediately adjacent to the ori beta region, also replicated in these two assays.

Animals↗