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W C Nierman

Publications and source records attributed to W C Nierman.

33 records · Page 2Linked to original sources
PubMed1994

Chromosome assignment of human brain expressed sequence tags (ESTs) by analyzing fluorescently labeled PCR products from hybrid cell panels.

Sixty-three human brain cDNA sequences were newly assigned to individual human chromosomes. Ten of these were subregionally localized, and one was also mapped in the mouse genome. Four previously reported assignments were refined. PCR primers were designed from expressed sequence tags (ESTs) and tested for specific amplification from human genomic DNA. DNA was then amplified, often in multiplexed PCR reactions, using DNA from somatic cell hybrid mapping panels as templates. The amplification products were identified using an automated fluorescence detection system. Chromosomal assignments were made by discordancy analysis. Thirteen newly localized cDNAs exhibited homology to previously reported sequences. EST01471 was shown to correspond to human microtubule-associated protein 1B (MAP1B), confirming the previous mapping of this gene to human chromosome 5. Other genes tentatively assigned to chromosomes based on these results were a component of the signal peptide receptor of the endoplasmic reticulum (EST00745) and a cyclic AMP-regulated phosphoprotein (EST01041) on chromosome 1, a protein phosphatase 2A 55-kDa regulatory subunit (EST01650) on chromosome 4, an NAD(P) transhydrogenase (EST01744) on chromosome 5, ribosomal proteins L1a or L1b (EST01627) and L18a (EST01583), a brain transcription factor (BF-1, EST00795) on chromosome 14, a milk fat globule membrane-related protein (EST01678) on chromosome 15, a putative peptide initiation factor (EST00675) on chromosome 17, thiosulfate sulfurtransferase (TST) on chromosome 22, and moesin (MSN) (EST00896) and a human equivalent of rat spot 14 (S14) (EST00887) on Xp11-->cen and Xpter-->p21.3, respectively.

Animals↗
PubMed1993

Chromosomal assignment of the human thrombin receptor gene: localization to region q13 of chromosome 5.

A functional thrombin receptor (TR) structurally related to other members of the seven-transmembrane receptor family has been isolated from diverse cellular types intimately involved in the regulation of the thrombotic response. This receptor recapitulates many of the previously identified sequelae of thrombin-mediated cell activation phenomenon, and requires proteolytic cleavage for downstream effector-response coupling events. Using two complementary approaches, we have now completed the chromosomal assignment of the human thrombin receptor gene. Discordancy analysis of polymerase chain reaction products from a human-rodent hybrid cell mapping panel assigned the sequence to human chromosome 5 with no observed discordancies. Cytogenetic localization using fluorescence in situ hybridization on human metaphase chromosomes specifically localized the human TR gene to region q13 of chromosome 5, confirming its presence as a single-locus gene in the human genome. The chromosomal localization of the human TR gene is at or contiguous with the proximal breakpoint site identified in the majority of patients with the 5q- syndrome (dysmegakaryocytopoiesis and refractory anemia).

Chromosome Mapping↗
PubMed1992

Chromosomal assignment of 38 human brain expressed sequence tags (ESTs) by analyzing fluorescently labeled PCR products from hybrid cell panels.

We have localized 38 human brain cDNA sequences to individual human chromosomes. PCR primers were designed from expressed sequence tags and tested for specific amplification from human genomic DNA. The sizes of amplification products from DNA of somatic cell hybrid mapping panels were determined electrophoretically using an automated fluorescence detection system. Chromosomal assignments were made by discordancy analysis.

Animals↗
PubMed1983

In vitro transcription of Drosophila actin and 70,000-dalton heat shock protein genes.

Drosophila genomic DNAs containing a chromosomal locus 87C1 70,000-dalton heat shock protein gene, the locus 79B actin gene, and the 88F actin gene have been used as templates in an in vitro HeLa transcription system. RNA polymerase II-dependent transcription initiates from specific sites on the heat shock protein gene and the 79B actin gene. The locations of the transcription start sites were determined by two types of experiments: sizing of RNA runoff transcripts and S1 nuclease mapping of the 5' terminus of the in vitro transcripts. Transcription initiates at or near the in vivo initiation site of the heat shock protein gene and initiates at or near a site 14 nucleotides downstream of the in vivo start site of the 79B actin gene. The addition of the 79B actin, 88F actin, or heat shock protein templates to a HeLa extract transcription reaction precluded the transcription of a second template subsequently added. The exclusion occurs rapidly, within 15 s, is not dependent on transcription, and is only partially resistant to high concentrations of the second added template. We propose that stable protein-promoter complexes play an important role in maintaining exclusive transcription of the first template added in vitro.

Actins↗
PubMed1981

A direct effect of guanosine tetraphosphate on pausing of Escherichia coli RNA polymerase during RNA chain elongation.

The effect of the regulatory nucleotide ppGpp on transcription by Escherichia coli RNA polymerase in vitro has been studied using bacteriophage T7 and T3 DNAs as templates. We have previously described the development of transcriptional test systems using these templates that can sensitively detect changes in promotor or terminator recognition of an RNA polymerase (Wiggs, J.L., Bush, J.W., and Chamberlin, M.J. (1979) Cell 16, 97-109) or changes in the rate of any of the major steps of the transcription cycle (Chamberlin, M.J., Nierman, W.C., Wiggs, J.L., and Neff, N. (1979) J. Biol. Chem. 254, 10061-10069). Using these procedures we fail to detect any substantial alteration by ppGpp of the normal interaction of E. coli RNA polymerase with the several T7 major and minor promoter sites or of the rate of productive RNA chain initiation at either T7 promoter A1 (ATP start) or A2 (GTP start). However, at physiologically relevant concentrations (KI approximately 50 microM), ppGpp significantly lowers the overall elongation rate of T7 RNA chains, leading to a substantial reduction in the overall rate of RNA synthesis. Inhibition of transcriptional elongation does not appear to be competitive with the ribonucleoside triphosphate substrates. Furthermore, ppGpp does not inhibit chain elongation during transcription of the synthetic polynucleotide templates poly[d(A-T)] or poly[d(A-G):d(C-T)]. We conclude that ppGpp interacts directly with some site on RNA polymerase other than one of the sites used for substrate binding. Furthermore, the inhibition must depend on specific DNA sequences present in T7 DNA, but not in poly[d(A-T)] or poly[d(A-G):d(C-T)]. Analysis of T7 transcripts formed in the presence and absence of ppGpp by gel electrophoresis reveals that in the latter instance there is enhanced pausing of the transcriptional elongation complex at specific sites on the template. It is likely that the enzyme also pauses at such sequences in the absence of ppGpp, but for a far briefer time (Kassavetis, G.A., and Chamberlin, M.J. (1981) J. Biol. Chem. 256, 2777-2786). Thus, ppGpp appears to slow transcriptional elongation by binding to RNA polymerase and altering its structure in a manner that impedes passage of the enzyme through certain DNA sequences. The presence of similar transcriptional barriers in rRNA operons activated by the presence of ppGpp could lead selectively to large reductions in the rate of rRNA synthesis in vivo.

DNA, Viral↗
PubMed1980

The effect of low substrate concentrations on the extent of productive RNA chain initiation from T7 promoters A1 and A2 by Escherichia coli RNA polymerase.

The extent of productive RNA chain initiation in vitro by Escherichia coli RNA polymerase holoenzyme from the bacteriophage T7 A1 and A2 promoters on purified T7 DNA templates has been investigated at very low concentrations of the ribonucleoside triphosphate substrates. As the concentration of ribonucleoside triphosphates in the reaction is decreased from 10 to 1 micro M, the extent of productive RNA chain initiation at these promoter sites drops precipitously at about 3 micro M. At 1 micro M substrate concentration, productive chain initiation from the A1 promoter does not occur even after extended incubation although the dinucleoside tetraphosphate pppApU is produced at a significant rate under these conditions. The reason for the inability of RNA polymerase to carry out productive RNA chain initiation at 1 micro M substrate concentration is not yet understood. The phenomenon is not due to substrate consumption, enzyme inactivation, or a requirement for a nucleoside triphosphatase activity in the reaction. The possibility is raised that there are additional nucleoside triphosphate binding sites on E. coli RNA polymerase which play some role in the process of productive RNA chain initiation.

DNA, Bacterial↗
PubMedDate not supplied

Chromosome painting and quantitative karyotyping of colon adenocarcinoma cell lines, DLD-1 and HCT-15.

Chromosome painting by fluorescence in situ hybridization (FISH) was used to examine abnormalities identified by G-banding in colon cancer lines, DLD-1 (ATCC CCL 221) and HCT-15 (CCL 225). DNA libraries from chromosomes comprising these abnormalities (i.e., N2, N8, N11, N16, N17 and N20) were used to prepare paint probes by PCR amplification. Of these paint probes, N2 and N8 exhibited additional chromosome-specific hybridization signals on centromeres that were also useful as a marker for chromosome identification. Results from FISH-painting and G-band analysis were consistent and permitted our quantitative analysis on karyotype evolution in vitro. In DLD-1, predominant cells having trisomic N20 in early passages were replaced by others with disomic N20 in late passages resulting in the trisomic 2p13-23 segment as the only deviation from the diploid content. In HCT-15, predominant cells having t(16;16) and double Y chromosome copies in early passages were replaced by those bearing the paired N16 and single Y chromosome in later passages. Thus cultures changed from the predominant hyperdiploidy to the sole pseudodiploidy with increased number of normal chromosomes.

Adenocarcinoma↗