scholarly journals DNA strand specificity for UV-induced mutations in mammalian cells.

1989 ◽  
Vol 9 (3) ◽  
pp. 1277-1283 ◽  
Author(s):  
H Vrieling ◽  
M L Van Rooijen ◽  
N A Groen ◽  
M Z Zdzienicka ◽  
J W Simons ◽  
...  
Keyword(s):  
Dna Replication ◽  
Base Pair ◽  
Mammalian Cells ◽  
Chinese Hamster ◽  
Parental Line ◽  
Induced Mutations ◽  
Hprt Gene ◽  
V79 Cells ◽  
H1 Cells ◽  
Double Base

The influence of DNA repair on the molecular nature of mutations induced by UV light (254 nm) was investigated in UV-induced hprt mutants from UV-sensitive Chinese hamster cells (V-H1) and the parental line (V79). The nature of point mutations in hprt exon sequences was determined for 19 hprt mutants of V79 and for 17 hprt mutants of V-H1 cells by sequence analysis of in vitro-amplified hprt cDNA. The mutation spectrum in V79 cells consisted of single- and tandem double-base pair changes, while in V-H1 cells three frameshift mutations were also detected. All base pair changes in V-H1 mutants were due to GC----AT transitions. In contrast, in V79 all possible classes of base pair changes except the GC----CG transversion were present. In this group, 70% of the mutations were transversions. Since all mutations except one did occur at dipyrimidine sites, the assumption was made that they were caused by UV-induced photoproducts at these sites. In V79 cells, 11 out of 17 base pair changes were caused by photoproducts in the nontranscribed strand of the hprt gene. However, in V-H1 cells, which are completely deficient in the removal of pyrimidine dimers from the hprt gene and which show a UV-induced mutation frequency enhanced seven times, 10 out of 11 base pair changes were caused by photoproducts in the transcribed strand of the hprt gene. We hypothesize that this extreme strand specificity in V-H1 cells is due to differences in fidelity of DNA replication of the leading and the lagging strand. Furthermore, we propose that in normal V79 cells two processes determine the strand specificity of UV-induced mutations in the hprt gene, namely preferential repair of the transcribed strand of the hprt gene and a higher fidelity of DNA replication of the nontranscribed strand compared with the transcribed strand.

DNA strand specificity for UV-induced mutations in mammalian cells

1989 ◽  
Vol 9 (3) ◽  
pp. 1277-1283
Author(s):  
H Vrieling ◽  
M L Van Rooijen ◽  
N A Groen ◽  
M Z Zdzienicka ◽  
J W Simons ◽  
...  
Keyword(s):  
Dna Replication ◽  
Base Pair ◽  
Mammalian Cells ◽  
Chinese Hamster ◽  
Parental Line ◽  
Induced Mutations ◽  
Hprt Gene ◽  
V79 Cells ◽  
H1 Cells ◽  
Double Base

The influence of DNA repair on the molecular nature of mutations induced by UV light (254 nm) was investigated in UV-induced hprt mutants from UV-sensitive Chinese hamster cells (V-H1) and the parental line (V79). The nature of point mutations in hprt exon sequences was determined for 19 hprt mutants of V79 and for 17 hprt mutants of V-H1 cells by sequence analysis of in vitro-amplified hprt cDNA. The mutation spectrum in V79 cells consisted of single- and tandem double-base pair changes, while in V-H1 cells three frameshift mutations were also detected. All base pair changes in V-H1 mutants were due to GC----AT transitions. In contrast, in V79 all possible classes of base pair changes except the GC----CG transversion were present. In this group, 70% of the mutations were transversions. Since all mutations except one did occur at dipyrimidine sites, the assumption was made that they were caused by UV-induced photoproducts at these sites. In V79 cells, 11 out of 17 base pair changes were caused by photoproducts in the nontranscribed strand of the hprt gene. However, in V-H1 cells, which are completely deficient in the removal of pyrimidine dimers from the hprt gene and which show a UV-induced mutation frequency enhanced seven times, 10 out of 11 base pair changes were caused by photoproducts in the transcribed strand of the hprt gene. We hypothesize that this extreme strand specificity in V-H1 cells is due to differences in fidelity of DNA replication of the leading and the lagging strand. Furthermore, we propose that in normal V79 cells two processes determine the strand specificity of UV-induced mutations in the hprt gene, namely preferential repair of the transcribed strand of the hprt gene and a higher fidelity of DNA replication of the nontranscribed strand compared with the transcribed strand.

Sequence specificity of point mutations induced during passage of a UV-irradiated shuttle vector plasmid in monkey cells

1986 ◽  
Vol 6 (1) ◽  
pp. 277-285 ◽  
Author(s):  
J Hauser ◽  
M M Seidman ◽  
K Sidur ◽  
K Dixon
Keyword(s):  
Dna Synthesis ◽  
Hot Spots ◽  
Mammalian Cells ◽  
Shuttle Vector ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Mutation Induction ◽  
Sequence Specificity ◽  
Induced Mutations ◽  
Double Base

A simian virus 40-based shuttle vector was used to characterize UV-induced mutations generated in mammalian cells. The small size and placement of the mutagenesis marker (the supF suppressor tRNA gene from Escherichia coli) within the vector substantially reduced the frequency of spontaneous mutations normally observed after transfection of mammalian cells with plasmid DNA; hence, UV-induced mutations were easily identified above the spontaneous background. UV-induced mutations characterized by DNA sequencing were found primarily to be base substitutions; about 56% of these were single-base changes, and 17% were tandem double-base changes. About 24% of the UV-induced mutants carried multiple mutations clustered within the 160-base-pair region sequenced. The majority (61%) of base changes were the G . C----A . T transitions; the other transition (A . T----G . C) and all four transversions occurred at about equal frequencies. Hot spots for UV mutagenesis did not correspond to hot spots for UV-induced photoproduct formation (determined by a DNA synthesis arrest assay); in particular, sites of TT dimers were underrepresented among the UV-induced mutations. These observations suggest to us that the DNA polymerase(s) responsible for mutation induction exhibits a localized loss of fidelity in DNA synthesis on UV-damaged templates such that it synthesizes past UV photoproducts, preferentially inserting adenine, and sometimes misincorporates bases at undamaged sites nearby.

scholarly journals Mapping an origin of DNA replication at a single-copy locus in exponentially proliferating mammalian cells.

1990 ◽  
Vol 10 (9) ◽  
pp. 4685-4689 ◽  
Author(s):  
L T Vassilev ◽  
W C Burhans ◽  
M L DePamphilis
Keyword(s):  
Dna Replication ◽  
Mammalian Cells ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Simian Virus 40 ◽  
Single Copy ◽  
Simian Virus ◽  
Metabolic Inhibitors ◽  
Ovary Cells ◽  
Cellular Origin

A general method for determining the physical location of an origin of bidirectional DNA replication has been developed recently and shown to be capable of correctly identifying the simian virus 40 origin of replication (L. Vassilev and E. M. Johnson, Nucleic Acids Res. 17:7693-7705, 1989). The advantage of this method over others previously reported is that it avoids the use of metabolic inhibitors, the requirement for cell synchronization, and the need for multiple copies of the origin sequence. Application of this method to exponentially growing Chinese hamster ovary cells containing the nonamplified, single-copy dihydrofolate reductase gene locus revealed that DNA replication begins bidirectionally in an initiation zone approximately 2.5 kilobases long centered about 17 kilobases downstream of the DHFR gene, coinciding with previously described early replicating sequences. These results demonstrate the utility of this mapping protocol for identifying cellular origins of replication and suggest that the same cellular origin is used in both the normal and the amplified DHFR locus.

scholarly journals Structural Changes in HPRT Gene of V79 Cells After Irradiation With Heavy Ions—Immediate and Delayed Effects

2021 ◽  
Vol 8 ◽  
Author(s):  
Pavel Bláha ◽  
Igor V. Koshlan ◽  
Nataliya A. Koshlan ◽  
Yulia V. Bogdanova ◽  
Daria V. Petrova ◽  
...  
Keyword(s):  
Heavy Ions ◽  
Mammalian Cells ◽  
Structural Changes ◽  
Chinese Hamster ◽  
Sharp Decrease ◽  
High Energy ◽  
Partial Deletion ◽  
V79 Cells ◽  
Delayed Effects ◽  
Radiobiological Effects

The radiobiological effects of accelerated ions with high charge and high energy (HZE) on mammalian cells and their propagation in time are still not sufficiently explained and attract great deal of attention. This work aims to compare the immediate and delayed effects with emphasis on the latter. As shown by our group, the dependence of mutant fraction on expression time after irradiation may have interesting, non-monotonic, character depending on LET (linear energy transfer) of the used heavy ions. We speculate that this phenomenon may occur due to the induced genomic instability. Another area of our research is the study of the DNA structural changes in these mutants induced at different expression times. Chinese hamster V79 cells were irradiated with accelerated ions 11B, 18O, 20Ne, and gamma radiation. The LET was ranging from 0.23 keV/μm of 60Co gamma rays up to 136 keV/μm of 20Ne ions. DNA of unique HPRT mutants was isolated, concentration measured, HPRT exons amplified, and analyzed at several different time points, up to about 40 days, after exposure. Over 1200 HPRT mutants were analyzed for deletions of exons and sorted into three main categories: partial deletion, PD—with deletion of one to eight exons; total deletions, TD—with all nine exons deleted; and no deletions—no change in the HPRT structure observed. In general, the number of samples with partial deletion was increasing with LET of the used radiation, suggesting that higher energy deposition to the cell nucleus is more likely to cause larger structural changes. In the case of total deletions, increase in their number with LET was observed up to LET ∼115 keV/μm followed by a sharp decrease. The samples were also analyzed for the distribution of deletions, in particular exons at various expression times, the so-called mutational patterns. Hypothesis of the mechanisms behind observed phenomena is given, and possible implications for further research are discussed.

Fish oil slows S phase progression and may cause upstream shift of DHFR replication origin ori-β in CHO cells

2002 ◽  
Vol 283 (4) ◽  
pp. C1009-C1024 ◽  
Author(s):  
Nawfal W. Istfan ◽  
Zhi-Yi Chen ◽  
Sybille Rex
Keyword(s):  
Dna Replication ◽  
Mammalian Cells ◽  
Replication Origin ◽  
Dna Analysis ◽  
Corn Oil ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
S Phase ◽  
Replication Initiation ◽  
Temporal Organization

Fish oils (FOs) have been noted to reduce growth and proliferation of certain tumor cells, effects usually attributed to the content of polyunsaturated fatty acids of the n–3 family, which are thought to modulate cellular signaling pathways. We investigated the influence of FO on cell cycle kinetics of cultured Chinese hamster ovary cells. Exponentially growing cells were labeled with 5-bromo-2′-deoxyuridine (BrdU) and analyzed by flow cytometry after 5-day treatment with exogenous fat. Bivariate BrdU-DNA analysis indicated slower progression through S phase and thus longer S phase duration time in FO- but not corn oil-treated or control cells. We hypothesize that FO treatment might interfere with spatial/temporal organization of replication origins. Therefore, we mapped the well-characterized replication origin ori-β downstream of the dihydrofolate reductase gene with the nascent strand length assay. Three DNA marker segments with known positions relative to this origin were amplified by PCR. By quantitatively assessing DNA length of the fragments in all fractions containing these markers, the location of ori-β was established. In control or corn oil-treated cells, the location of ori-β was consistent with previous studies. However, in FO-treated cells, DNA replication appears to start from a new site located farther upstream from ori-β, suggesting a different replication initiation pattern. This study suggests novel mechanism(s) by which fats affect cell proliferation and DNA replication in mammalian cells.

Correlation of gene expression and transformation frequency with the presence of an enhancing sequence in the transforming DNA

1984 ◽  
Vol 4 (2) ◽  
pp. 368-370
Author(s):  
P E Berg ◽  
W F Anderson
Keyword(s):  
Gene Expression ◽  
Dna Sequences ◽  
Base Pair ◽  
Mammalian Cells ◽  
Chinese Hamster ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Transformation Frequency ◽  
Hamster Strain ◽  
Sarcoma Virus

The transformation frequency of cultured mammalian cells is increased 10- to 100-fold when certain DNA sequences are present in the transforming DNA. We wanted to determine whether enhancers, which stimulate gene expression, can cause this phenomenon. Three plasmids, each containing a galactokinase K (galK) gene, were used to transform galK- Chinese hamster cells. One plasmid has no enhancer, another has the simian virus 40 (72-base-pair repeat) enhancer, and the third has the Harvey sarcoma virus (73-base-pair repeat) enhancer. The presence of either enhancer significantly increased the appearance of GalK+ colonies. Galactokinase transient assays in this Chinese hamster strain in the presence of the same plasmids demonstrated an increase in GalK enzyme levels when either enhancer was present. These data indicate that there is a strong correlation between galK expression and transformation frequency that is dependent on the presence of an enhancer in the transforming DNA.

DNA sequence analysis of 1-nitrosopyrene-induced mutations in the hprt gene of Chinese hamster ovary cells

1992 ◽  
Vol 13 (5) ◽  
pp. 819-825 ◽  
Author(s):  
Ronald K. Newton ◽  
Robert A. Mittelstaedt ◽  
Mugimane.G. Manjanatha ◽  
Robert.H. Heflich
Keyword(s):  
Sequence Analysis ◽  
Dna Sequence ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Dna Sequence Analysis ◽  
Induced Mutations ◽  
Hprt Gene ◽  
Ovary Cells

scholarly journals Correlation of gene expression and transformation frequency with the presence of an enhancing sequence in the transforming DNA.

1984 ◽  
Vol 4 (2) ◽  
pp. 368-370 ◽  
Author(s):  
P E Berg ◽  
W F Anderson
Keyword(s):  
Gene Expression ◽  
Dna Sequences ◽  
Base Pair ◽  
Mammalian Cells ◽  
Chinese Hamster ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Transformation Frequency ◽  
Hamster Strain ◽  
Sarcoma Virus

The transformation frequency of cultured mammalian cells is increased 10- to 100-fold when certain DNA sequences are present in the transforming DNA. We wanted to determine whether enhancers, which stimulate gene expression, can cause this phenomenon. Three plasmids, each containing a galactokinase K (galK) gene, were used to transform galK- Chinese hamster cells. One plasmid has no enhancer, another has the simian virus 40 (72-base-pair repeat) enhancer, and the third has the Harvey sarcoma virus (73-base-pair repeat) enhancer. The presence of either enhancer significantly increased the appearance of GalK+ colonies. Galactokinase transient assays in this Chinese hamster strain in the presence of the same plasmids demonstrated an increase in GalK enzyme levels when either enhancer was present. These data indicate that there is a strong correlation between galK expression and transformation frequency that is dependent on the presence of an enhancer in the transforming DNA.

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