A brief history of the discovery of sister chromatid exchanges.
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
J H Taylor
Publications and source records attributed to J H Taylor.
Origins of replication and gene regulation.
Eukaryotic chromosomes appear to consist of many replicons, the time of replication of which is probably controlled by specific origins. However, plasmids without specific eukaryotic origins may also replicate in some cells when injected into nuclei or transferred during transformation. The efficiency and the mechanisms of their initiation are still uncertain. A number of reports are cited which indicate that natural eukaryotic DNAs initiate their replication from specific origins. The nature of these origins are known in only a few instances and no general conclusions can yet be given about the nucleotide sequences involved. Short dispersed repeats of the Alu type appear to function as origins since they enhance the efficiency of replication of vector plasmids in Xenopus eggs. Certain sequences from a variety of eukaryotic DNAs also enhance the replicative potential of plasmids in yeast cells. The common features of such initiators or enhancers is uncertain. If dispersed repeats are origins in mammalian chromosomes, the number appears to be excessive. Either only a subset are functional, or the functional ones are only suborigins in larger replicons in which master origins (not yet isolated) function in the regulation of the timing of replication. Evidence is cited which indicates that the regulation of the time of replication of a gene or gene cluster is part of a regulatory system that makes the DNA available for transcription or leaves it in an inactive state. About one-half the DNA in mammalian cells is replicated in the first half of S phase (SE). After a brief pause in mid-S phase, the remainder of the DNA is replicated in what is designated late S (SL). The fractions replicated in SE and SL may vary in other phylogenetic groups, but wherever division of differentiated cells occurs such fractions are likely to be found. The following hypothesis is proposed. The DNA replicated in SL is suppressed in transcription, if it has the appropriate promoter regions, because the newly replicated DNA is complexed with proteins that suppress transcription. These proteins are only available during SL. Those genes replicated in SE are complexed with a different set of proteins which leave the promoter regions open for transcription when the appropriate regulatory molecules are available. In this way an inactive state or potentially active state can be transmitted from one cell generation to the next. Evidence is cited which indicates that genes which are active in all cells at some stage in the cell cycle are replicated in SE.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
Controlled study of 16,16-dimethyl-trans-delta 2 prostaglandin E1 methyl ester vaginal pessaries prior to suction termination of first trimester pregnancies.
A pessary containing the prostaglandin analogue, 16,16-dimethyl-trans-delta 2 prostaglandin E1 methyl ester (Cervagem) was inserted into the vagina of primigravid women 2 h before suction termination of pregnancy. The cervix was softer, easier to dilate and more widely dilated at the start of the procedure than in a control group of women who received placebo pessaries containing the vehicle alone. Intra-operative blood loss was diminished. The incidence of pre- and post-operative abdominal cramps was increased in the Cervagem-treated group. No gastrointestinal side-effects were noted.
Abortion, Induced↗
The quantitation of sister chromatid exchanges in lymphocytes of cancer patients at intervals after cytotoxic chemotherapy.
Venous blood was taken from patients with cancer, prior to and up to 42 days after the administration of cytotoxic chemotherapy. Lymphocytes were stimulated to divide in vitro, and examined for sister chromatid exchanges (SCEs). Cyclophosphamide rapidly increased the frequency of SCE, which returned to approximately double the control value 24 hr after administration. The remaining SCEs disappeared more slowly. There was a positive correlation between the dose of drug and the frequency of SCE measured immediately and 4 hr, 20 hr and 21 days after treatment. As patients received successive courses of treatment the number of SCEs generally increased from about 0.14 to 0.25 per chromosome. After this, further chemotherapy was often less effective in inducing them. The presence of SCEs in peripheral blood lymphocytes may be a useful indicator for the occurrence and persistence of alkylating metabolites, residual damage in the DNA and individual responses of patients to a standard regimen.
Adult↗
Induction of sister chromatid exchanges by 5-fluorodeoxycytidine: correlation with DNA methylation.
Chinese hamster ovary cells grown in medium containing 10 microM 5-fluorodeoxycytidine for forty-eight hours were found to have up to 5% of the deoxycytidine residues of the DNA substituted by this analog. Cytological studies of these cells showed that the incorporated 5-FdC caused a two-fold increase of sister chromatid exchanges (SCE) over the control level. However, 5-FdC was capable of inducing SCE only after it had been present in the cell for at least two cycles of DNA synthesis. This is in contrast to several other chemicals that we have tested which induced SCE immediately after the first DNA synthesis. We consider the possibility that the delayed effect may be related to hypomethylation of cytosine in the newly replicated DNA.
Animals↗
Sister chromatid exchanges in human lymphocytes treated with combinations of cytotoxic drugs.
Lymphocytes from peripheral blood of either 'normal' donors or patients with cancer were stimulated to divide in culture medium containing 5-bromo-2'-deoxyuridine. During 74 hr of incubation, the cells were exposed to single agents or to permutations of two combinations of chemotherapeutic drugs, namely MOPP or cyclophosphamide and methotrexate. Only mustine and cyclophosphamide (activated and not activated) increased the frequency of sister chromatid exchanges (SCE). Neither vincristine, procarbazine or prednisolone with mustine, nor methotrexate with cyclophosphamide altered the number of SCEs expected from the use of mustine or cyclophosphamide alone. There was no difference in the response of cells from cancer patients and 'normal' subjects to the drugs. If the drugs of these two regimens do interact with one another to enhance the amount of subcellular damage, this is not manifested by changes in the number of SCEs in human lymphocytes in vitro.
Adult↗
Sister chromatid exchanges in human lymphocytes exposed to single cytotoxic drugs in vivo or in vitro.
Venous blood was taken from apparently healthy volunteers and patients with cancer, the latter both before and at intervals after treatment with single cytotoxic drugs. Cells from untreated individuals were exposed to a range of concentrations of drugs in culture medium. Chlorambucil, treosulfan and cyclophosphamide (activated by hepatic microsomes) significantly increased the numbers of SCEs, both in vitro and in the lymphocytes of patients. While methotrexate and 5-fluorouracil had no effect, bleomycin slightly increased the number of SCEs, but only in vitro. Although the in vitro dose-effect relationship indicated which drugs would increase the frequency of SCE in vivo, the magnitude of the response tended to be overestimated. When patients were treated with drugs the frequency of SCE increased, then declined with time. Though this complicates the quantitative relationship between dose and damage, SCEs may be useful to monitor the effects of alkylating agents on normal tissues.
Adult↗
Dissection of a replication origin of Xenopus DNA.
A previously cloned 503-base pair (bp) EcoRI segment of genomic DNA from Xenopus laevis selected for enhancement of replication of its vector plasmid was moved to the EcoRI site of pBR322. This plasmid designated pJCC31 and five other clones, which were made by cleaving the 503-bp segment in relation to a dispersed repeated sequence and subcloning, were compared with pBR322 for replication by microinjection into Xenopus eggs. The replication measured by incorporation of a 32P-labeled nucleotide as well as semiconservative segregation and dilution of N6-methyladenine at the EcoRI sites showed pJCC31 to be about 15 times as efficient as pBR322. The next most efficient subclone, pJCC31-2, contains an insert with a complete 320-bp dispersed repeated sequence bracketed by an 8-bp direct repeat. This observation, along with our previous report that repeated sequences of the Alu family in the human genome enhanced replication of the vector plasmid nearly as much as that of the presumptive Xenopus origin, leads to the hypothesis that members of a subset of the short dispersed repeated sequences in vertebrates function as origins for chromosomal replication. Preliminary studies also show that the presumptive Xenopus origin contains a RNA polymerase promoter that increases the transcription of the plasmid when it is microinjected into Xenopus oocytes.
Animals↗
Distribution of 5-methylcytosine in the DNA of somatic and germline cells from bovine tissues.
Genomic DNA of calf thymus contains 1.5 times as much 5-methylcytosine as similar sperm DNA, but the major EcoRI repeat fragment from satellite I of thymus contains ten times as much 5-methylcytosine as the corresponding fragment from sperm DNA. Restriction enzyme analyses of the total DNA and the satellite I fragment show that three HpaII sites in the fragment are completely unmethylated in sperm but fully methylated in thymus DNA. Under-methylation of many sites in the satellite DNAs can probably account for the lower level of methylation of sperm DNA rather than hemimethylation as previously suggested. These results are also discussed in relation to maintenance and de novo (initiation-type) methylases.
5-Methylcytosine↗
Further studies on the subunit structure and oligosaccharide moiety of human enterokinase.
Highly purified human enterokinase was found by SDS-polyacrylamide gel electrophoresis to contain three heavily glycosylated subunits of apparent molecular masses 54 000, 102 000 and 140 000. The smallest subunit contained the active site serine residue and the oligosaccharide chains appear to be N-glycosidically linked as inferred from their stability to mild alkaline hydrolysis. Lectin affinity chromatography was used to separate sub-populations of the enzyme, the major one of which appeared to contain terminal alpha -linked N-acetyl galactosamine. Despite the presence of this sugar, no anti-A response was elicited in rabbits immunized with this sub-population. However, this sub-population did bind rabbit antibody directed against human blood group A substance, suggesting the presence of an "A-like" determinant. Studies with immobilized rabbit anti-human blood group A IgG suggest that there is no correlation between the blood group of an individual and the antigenic determinants on the enterokinase produced by the enterocytes of that individual. The study of the molecular properties of this important enzyme may give insights into pathological conditions with which it is linked.
ABO Blood-Group System↗
Cloning of an origin of DNA replication of Xenopus laevis.
DNA fragments of Xenopus laevis, the African frog, were cloned in the EcoRI site of the Escherichia coli plasmid pACYC189 and tested for ability to initiate and complete replication of the recombinant plasmid when injected into unfertilized eggs of X. laevis. After measurement of the [3H]-thymidine incorporation per egg for a number of recombinant plasmids, pSW14 and pSW9, which respectively contain a small segment (550 base pairs) and several kilobases of frog DNA, were selected for more extensive analysis. In spite of the small size of the segment in pSW14, it incorporates in 2 hr at least 3 times as much labeled thymidine as either pSW9 or the vector alone. The DNA synthesis in pSW14 was shown to be replication rather than repair synthesis, based on a buoyant density shift of the product when iododeoxyuridine was used for labeling. To determine the number of replications of pSW14, a novel method was employed. Because pSW14 is a head-to-head dimer of the vector with the Xenopus fragment inserted at an EcoRI site, the plasmid has three methylatable sites--two bracketing the Xenopus fragment and one opposite the fragment. By cotransformation of E. coli with pSW14 and pBR322 containing the EcoRI methylase gene, supercoiled pSW14 was methylated and injected into eggs with [3H]thymidine. Disappearance of modified EcoRI sites by semiconservative replication was followed by measuring the sensitivity to EcoRI endonuclease over time. The results showed that about 50% of the labeled, supercoiled DNA recovered from eggs after 4 hr was sensitive to EcoRI digestion, which indicates that most of the DNA that incorporated [3H]thymidine had replicated twice during the 4 hr in the unfertilized eggs of X. laevis. We conclude that pSW14 has a functional origin in the Xenopus DNA segment.
Animals↗
The effects of X rays on DNA synthesis in synchronized Chinese hamster ovary cells.
Explore the source record for details and available documents.
Animals↗
Initiation of DNA replication in chromosomes of Chinese hamster ovary cells.
The initiation of DNA replication and the subsequent chain elongation were studied using Chinese hamster ovary cells synchronized at the beginning of S phase. The cells were synchronized by a combination of mitotic selection and treatment with 5-fluorodeoxyuridine (FdU). The use of this drug at a concentration of 10(-5) M was found to effectively prevent the leakage of cells into S phase. Reversal of the FdU block by supplying thymidine resulted in the synchronous onset of initiation at multiple sites in each cell. The length of the nascent chains, as determined by autoradiography and velocity sedimentation in alkaline gradients, increased linearly with time during the first twenty minutes of S phase after release. We applied these procedures to study the effects of the length of an FdU block on the number of functional origins per cell, the rate of chain growth, and the rate of DNA synthesis per cell following reversal of the block. Although no change was noted in the rate of DNA synthesis in cells held at the beginning of S phase from 10.5 to 24 h after division, the rate of chain growth decreased from 0.94 to 0.28 microns per min. This decrease indicated that the number of functional origins increased markedly with length of FdU block. The calculated number of utilized origins per cell increased from 1,900 to 5,700. We also presented arguments that 1,900 origins per cell represents the approximate number of origins utilized by any cell held at the beginning of S phase for less than 10.5 h after division.
Animals↗
Effect of ultraviolet irradiation on eggs and larvae on the northern anchovy, Engraulis mordax, and the Pacific mackerel, Scomber japonicus, during the embryonic stage.
Explore the source record for details and available documents.
Animals↗
Fire, disease and water in Ottawa: an introduction.
Explore the source record for details and available documents.
Canada↗
Disease, fire and water in the nineteenth century city: a bibliography.
Explore the source record for details and available documents.
Canada↗
Actinomycin D inhibits initiation of DNA replication in mammalian cells.
Mammalian (Chinese hamster ovary) cells were synchronized in the division cycle and blocked at the beginning of S phase with fluorodeoxyuridine. Traces of thymidine in the medium allowed cells to enter S phase and initiate DNA replication at some origins. For many hours after the traces of thymidine were depleted new sites for DNA replication accumulated in a small fraction of the DNA. However, these potential origins became active in bidirectional replication only when cells were released by adding [3H]thymidine to the medium. Lysis at 37 degrees C released most of the pulse-labeled DNA as linear double-stranded segments larger than Okazaki fragments and smaller than the unreplicated parental DNA. Release of the newly replicated DNA involves breakage of the template chains at or near each fork. The size of the fragments increased linearly with time of pulse labeling, but the efficiency of their release decreased. The excision could be prevented by lysis at 0 degrees C. When cells were treated with actinomycin D for 3--5 min before release, the new origins failed to function, but chain growth continued from those sites at which initiation had taken place before depletion of thymidine. We interpret these results to indicate that initiation at origins requires an actinomycin D-sensitive step, presumably RNA transcription, while chain elongation, which involves the formation of Okazaki pieces, is relatively insensitive to actinomycin D during growth over long intervals.
Cell Line↗
Methylation of DNA in early development: 5-methyl cytosine content of DNA in sea urchin sperm and embryos.
By separating formic acid hydrolysates with high pressure chromatography on an Aminex-10 column, we determined the ratio of 5-methyl cytosine to cytosine and other bases of DNA from sea urchin sperm and nuclei of embryos from early cleavage through pluteus stages. Contrary to several previous reports, we could not find any measurable changes in the methylation levels of embryonic nuclear DNAs at different stages of development. We also found no consistent differences between the methylation levels of sea urchin sperm and embryonic nuclei or the 5-methyl cytosine content of fish (Mugil cephalus) sperm and liver nuclei. While these measurements would not have detected subtle variations associated with differentiation, they would have indicated the gross changes previously reported for embryos or between sperm and somatic nuclei had those changes been present.
Animals↗