Biomedical subjects

C M Kane

Publications and source records attributed to C M Kane.

47 records · Page 3Linked to original sources

PubMed1988

Intrinsic sites of transcription termination and pausing in the c-myc gene.

We have studied transcription elongation and termination in the human c-myc gene. Transcription of c-myc gene sequences with purified mammalian RNA polymerase II revealed several sites of transcription termination and pausing in the vicinity of the exon 1-intron 1 junction. This region previously has been shown to block transcription elongation in vivo by nuclear run-on analysis (D. Bentley and M. Groudine, Nature [London] 321:702-706, 1986). These sites were recognized by purified RNA polymerase II, and we therefore designated them intrinsic sites of termination and pausing. Two of these sites cause termination of RNA polymerase III transcription as well. RNA polymerase II terminated transcription in a cluster of seven consecutive T residues in the nontranscribed strand and paused during transcription at three additional sites in this region. The intrinsic sites of transcription termination and pausing described here correspond closely to the 3' ends of transcripts synthesized in Xenopus oocytes injected with plasmids containing the c-myc termination region (D. Bentley and M. Groudine, Cell 53:245-256, 1988). This correspondence suggests that the intrinsic recognition of these termination and pause sites by purified RNA polymerase II may play a role in the transcription elongation block observed in vivo.

Chromosome Mapping↗

PubMed1988

The Amherst study of hospital marketing practices.

The study findings identify two major area of concern related to marketing and the direction the field is taking. First, health care marketing--though much discussed and expensive--is still in a stage of immaturity. As practiced in hospitals today, marketing is frequently synonymous with advertising. Second, marketing in health care will not ultimately be a "clone" of marketing practices in industry and retailing. Though hospitals are looking to those areas for knowledge and expertise, the unique character of the product will cause health care marketing to evolve into a practice uniquely different from that in other industries.

Data Collection↗

PubMed1987

Identification of intrinsic termination sites in vitro for RNA polymerase II within eukaryotic gene sequences.

We have identified and mapped several DNA sequences within a human histone gene (H3.3) at which in-vitro transcription by highly purified RNA polymerase II is efficiently terminated. Since transcription in our system involves only RNA polymerase II acting on a linear DNA template, these sequences contain "intrinsic" termination signals recognized by the polymerase protein itself. The existence of such signals within a gene suggests that efficient antitermination systems probably exist for mammalian transcription units. Alternatively, there could be a high frequency of premature transcription termination, or "polarity" for genes such as H3.3. Intrinsic transcription termination sites in H3.3 are located in sequences of consecutive thymidylate residues (5 to 8 nucleotides) on the non-transcribed DNA strand (T-runs), from which it is likely that such T-runs are elements of the intrinsic termination signal for RNA polymerase II. However, transcription proceeds without significant termination through many similar T-runs, from which it follows that these intrinsic termination signals include other elements. Since transcription is also terminated efficiently at these sites when the transcript remains bound along its full length as a DNA-RNA hybrid, it is unlikely that formation of specific RNA secondary structures in the transcript is a general element of the intrinsic termination signal. Although DNA sequences downstream from the coding portion of the mouse beta-globin gene have been implicated as sites of transcription termination in vivo, these regions do not contain strong intrinsic termination signals, and transcription in vitro proceeds through these regions almost undiminished. Transcriptional termination in this region in vivo may depend on the presence of termination factors or other intracellular elements, and there may be multiple classes of DNA signals that control eukaryotic termination.

Animals↗

PubMed1987

Purified RNA polymerase II recognizes specific termination sites during transcription in vitro.

We have studied the ability of certain well-defined prokaryotic DNA sequences to act as specific termination signals for highly purified calf thymus RNA polymerase II. We used duplex DNA fragments modified to direct efficient and specific transcription of defined DNA templates to follow transcription with RNA polymerase alone in the absence of additional protein factors. Elongation of RNA chains by RNA polymerase II is processive through most DNA sequences. However, certain DNA sequences serve as effective "intrinsic" terminators for RNA polymerase II; in this they resemble the "rho-independent" terminators for the bacterial RNA polymerase. Several rho-independent bacterial terminators are also able to act as termination signals for RNA polymerase II. However, there is no apparent correlation between the efficiency of termination for the bacterial enzyme and that found for the calf thymus enzyme. One very efficient bacterial terminator (phage T7 early terminator) gives no termination with RNA polymerase II, and we have identified at least two sites that cause the eukaryotic enzyme to terminate but have no effect on transcription by the bacterial enzyme. Hence, the signals recognized as intrinsic termination sites for the two enzymes are substantially different. All of the sites that act as intrinsic terminators for RNA polymerase II contain a series of consecutive thymidine residues in the nontranscribed DNA strand (T-run), and the 3' end of the completed RNA normally lies within this sequence. It is plausible that the T-run is part of the signal for an RNA polymerase II termination site; however, there is no apparent correlation between the number of T residues and the efficiency of the terminator, suggesting that other sequence elements are required for, or modulate, termination. Several lines of evidence suggest that the formation of RNA secondary structures in the nascent transcript is not an essential element of the intrinsic RNA polymerase II termination signal.

Animals↗

PubMed1987

Acute cardiovascular responses to internal carotid artery occlusion during carotid endarterectomy and to the restoration of internal carotid flow.

Heart rate, central venous pressure, radial artery pressure and electrocardiograph were recorded in 14 patients undergoing carotid endarterectomy under general anaesthesia supplemented with fentanyl and halothane and muscle relaxation as required. Induction of anaesthesia was followed by a significant reduction in systolic arterial pressure and a rise in central venous pressure (CVP). Thereafter CVP did not vary significantly and heart rate did not change significantly at any stage. After internal carotid artery occlusion, mean systolic pressure at one minute, 143 (SD 17) mmHg, and three minutes, 160 (SD 27) mmHg, were both significantly higher than prior to occlusion, 132 (SD 17) mmHg (P less than 0.01 and P less than 0.002 respectively). Similar significant changes occurred in diastolic pressure after carotid occlusion (P less than 0.02 and P less than 0.002 respectively). The restoration of flow through the internal carotid artery in patients operated on without a shunt was associated with a significant reduction in mean systolic pressure. The mean systolic pressure at one minute, 145 (SD 20) mmHg, and three minutes, 135 (SD 19) mmHg, were both significantly lower than that before restoration of flow, 159 (SD 17) mmHg (P less than 0.02 and P less than 0.05 respectively). Changes in mean diastolic pressure in this group at these times, while in the same direction, were not significant. The observed hypertensive response to carotid occlusion may assist in preserving cerebral perfusion while the internal carotid artery is occluded, but may be hazardous for patients with ischaemic heart disease.

Aged↗

PubMed1985

Amino acid sequences of the carboxyl-terminal regions of rat plasma fibrinogen gamma A and gamma' chains.

Amino acid sequence analysis of human fibrinogen gamma' chains have shown that the larger size relative to the gamma A chains is due to differences at the COOH-terminus. As shown by DNA sequencing, these differences as well as those in rat fibrinogen gamma' and gamma A chains result from differential processing of the primary mRNA transcript. We isolated and sequenced carboxyl-terminal CNBr peptides from gamma' and gamma A chains. As predicted by DNA sequencing, the gamma A peptide has the carboxyl-terminal sequence Gly-Gly-Ser-Lys Gln-Val-Gly-Asp-Met-COOH. The gamma' peptide is identical through residue 6 and terminates with Ser-Val-Glu-His-Glu-Val-Asp-Val-Glu Tyr-Pro-COOH.

Amino Acid Sequence↗

PubMed1985

Preparation of functionally intact monomers by limited disulfide reduction of human plasma fibronectin dimers.

Most (90 to 95%) human plasma fibronectin (PFn) molecules exist as 450-kDa disulfide-rich dimers comprised of two major types of subunits (A, 220 kDa; B, 215 kDa) that are joined near the COOH terminus by two disulfide bonds. Smaller PFn species (Zone II; 190-235 kDa) consist mainly of monomers and/or a monomeric subunit joined covalently to a smaller peptide remnant presumably derived by proteolysis of a parent 450-kDa molecule. A relatively simple and selective method for preparing functionally active, partially reduced monomeric fibronectin subunits (PR-PFn) by limited and selective reduction of dimeric plasma fibronectin (PFn) has been developed. PR-PFn was prepared by incubating PFn in phosphate-buffered saline, pH 7.4, for 2 h at room temperature in the presence of 17 mM dithiothreitol (DTT). Following S-carboxymethylation or S-carboxyamidomethylation, the material was passed through a gelatin-Sepharose column and nonbinding material was discarded; gelatin-bound material was eluted using a 0 to 2 M KSCN gradient. Residual dimeric species (10-20%) could be separated from monomers in high yield by gel-sieving chromatography on a Sepharose 6B-Cl in the presence of a chaotropic salt, 0.3 M KSCN. Most new SH groups (74-81%) in that fraction of PR-PFn binding to gelatin were localized in proteolytic fragments containing the COOH terminus, thus suggesting that selective cleavage of the interchain disulfide bridges had taken place. The binding affinity of PR-PFn to gelatin- and fibrin-Sepharose was lower than that of dimeric PFn, but the same as that of Zone II PFn and other monomeric gelatin-binding proteolytic derivatives. PR-PFn also bound to heparin-Sepharose and promoted cell attachment and spreading. We conclude that PR-PFn monomers possess the same functional activities as those of the parent chains.

Chromatography, Affinity↗

PubMed1985

Studies on transcription of 3'-extended DNA templates by mammalian RNA polymerase II. Partial purification and characterization of a factor from HeLa cells that facilitates renaturation of the DNA template.

Transcription by purified mammalian RNA polymerase II in vitro leads to extensive formation of DNA-RNA hybrids between nascent RNA and the template DNA strand. This is especially clear during transcription of 3'-extended (dC-tailed) DNA templates where the nontranscribed DNA strand is progressively displaced as transcription proceeds [Kadesch, T. R., & Chamberlin, M. J. (1982) J. Biol. Chem. 257, 5286-5295]. Addition of small amounts of a HeLa cell extract to such a transcription system enhances renaturation of the template DNA and displacement of the nascent RNA, as measured by the sensitivity of the RNA to pancreatic ribonuclease. Using this latter assay, we have purified a protein factor (renaturase) 250-fold from HeLa cell extracts using chromatography on DEAE-cellulose, DNA-cellulose, and hydroxylapatite. Renaturase preparations facilitate complete renaturation of the template DNA duplex during transcription by RNA polymerase II and lead to concurrent displacement of the nascent RNA. Current preparations are free from all but traces of deoxyribonuclease or ribonuclease. The active component has a molecular weight of about 30000 as estimated by preparative density gradient sedimentation. We have examined the structure of transcribing RNA polymerase II complexes in the presence and absence of renaturase, using the electron microscope and the Williams polylysine technique [Williams, R. C. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 2311-2315]. In the presence of renaturase, the DNA template is fully renatured, and a ternary complex in which the nascent RNA is displaced during transcription is seen.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1982

Tissue-specific and species-specific monoclonal antibodies to avian red cell nuclear proteins.

In order to identify potential red cell-specific regulatory proteins and to define additional red cell-specific markers, we have isolated a series of hybridomas that produce monoclonal antibodies that react with nuclear preparations from avian red blood cells. Several antibodies have been well characterized for their tissue- and species-specific reactions by using solid-phase and protein-transfer radioimmunoassays as well as immunofluorescence. These antibodies should allow isolation and characterization of individual nuclear proteins that are tissue and species specific and may prove useful for the study of gene expression in the erythropoietic system. The majority of the well-characterized antibodies appear to have tissue-specific properties. However, three antibodies react with all tested chicken tissues; one of these reacts with multiple peptides in a pattern that varies qualitatively and quantitatively between the tissues. This may reflect a common protein domain or modification that is used in several different tissues for similar functions but is nevertheless present in an overall protein framework that is tissue specific. Because the major fraction of the hybridomas initially produced is tissue specific, we presume that the immune system selects for tissue-specific determinants. This property of the immune system may prove to be a useful general feature of this type of analysis.

Animals↗

PubMed1981

Purification and characterization of an apurinic/apyrimidinic endonuclease from HeLa cells.

An endodeoxyribonuclease from HeLa cells acting on apurinic/apyrimidinic (AP) sites has been purified to apparent homogeneity as judged by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The presence of Triton X-100 was necessary throughout the purification for stabilization and stimulation of activity. The endonuclease has an apparent native molecular weight of 32,000 determined by molecular sieving and an apparent subunit molecular weight of 41,000 as judged by its electrophoretic mobility in SDS-polyacrylamide gels. The activity has an absolute requirement for Mg2+ or Mn2+ and a broad pH optimum between 6.7 and 9.0 with maximal activity near pH 7.5. The enzyme has no detectable exonuclease activity, nor any endonuclease activity on untreated duplex or single-stranded DNA. It is inhibited by adenine, hypoxanthine, adenosine, AMP, ADP-ribose, and NAD+, but it is unaffected by caffeine, the pyrimidine bases, ADP, ATP, or NADH. The use of a variety of damaged DNA substrates provided no indication that the enzyme acts on other than AP sites. The enzyme appears to cleave AP DNA so as to leave deoxyribose-5-phosphate at the 5' terminus and a 3'-OH at the 3' terminus; it also removes deoxyribose-5-phosphate from AP DNA which has deoxyribose at the 3' terminus. Specific antibody has been produced in rabbits which interacts only with a 41,000-dalton protein present in the purified enzyme (presumably the enzyme itself), as well as with partially purified AP endonuclease fractions from human placenta and fibroblasts.

Animals↗

PubMed1980

Apurinic/apyrimidinic endonucleases in repair of pyrimidine dimers and other lesions in DNA.

The characteristics of the nicks (single-strand breaks) introduced into damaged DNA by Escherichia coli endonucleases III, IV, and VI and by phage T4 UV endonuclease have been investigated with E. coli DNA polymerase I (DNA nucleotidyltransferase). Nicks introduced into depurinated DNA by endonuclease IV or VI provide good primer termini for the polymerase, whereas nicks introduced into depurinated DNA by endonuclease III or into irradiated DNA by T4 UV endonuclease do not. This result suggests that endonuclease IV nicks depurinated DNA on the 5' side of the apurinic site, as does endonuclease VI, whereas endonuclease III has a different incision mechanism. T4 UV endonuclease also possesses apurinic endonuclease activity that generates nicks in depurinated DNA with low priming activity for the polymerase. The priming activity of DNA nicked with endonuclease III or T4 UV endonuclease can be enhanced by an additional incubation with endonuclease VI and, to a lesser extent, by incubation with endonuclease IV. These results indicate that endonuclease III and T4 UV endonuclease (acting upon depurinated and irradiated DNA, respectively) generate nicks containing apurinic/apyrimidinic sites at their 3' termini and that such sites are not rapidly excised by the 3' leads to 5' activity of DNA polymerase I. However, endonuclease IV or VI apparently can remove such terminal apurinic/apyrimidinic sites as well as cleave on the 5' side of the unnicked sites. These results suggest roles for endonucleases III, IV, and VI in the repair of apurinic/apyrimidinic sites as well as pyrimidine dimer sites in DNA. Our results with T4 UV endonuclease suggest that the incision of irradiated DNA by T4 UV endonuclease involves both cleavage of the glycosylic bond at the 5' half of the pyrimidine dimer and cleavage of the phosphodiester bond originally linking the two nucleotides of the dimer. They also imply that the glycosylic bond is cleaved before the phosphodiester bond.

Apurinic Acid↗