Amplification and direct nucleotide sequencing of cDNA from the lysate of low numbers of diploid human cells

Yang, Jialing; Maher, Veronica M.; McCormick, J. Justin

doi:10.1016/0378-1119(89)90121-2

Cited by 129 publications

(52 citation statements)

References 14 publications

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“…Firststrand cDNA was synthesized directly from mRNA in lysates of 100 to 500 cells, using the polymerase chain reaction as described previously (34). The amplified HPRT coding region was sequenced under the conditions recommended by the manufacturer (protocols for DNA sequencing with Sequenase version 2.0, U.S. Biochemical, Cleveland, Ohio).…”

Section: Methodsmentioning

confidence: 99%

Evidence from mutation spectra that the UV hypermutability of xeroderma pigmentosum variant cells reflects abnormal, error-prone replication on a template containing photoproducts.

et al. 1993

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Xeroderma pigmentosum (XP) variant patients are genetically predisposed to sunlight-induced skin cancer.Fibroblasts derived from these patients are extremely sensitive to the mutagenic effect of UV radiation and are abnormally slow in replicating DNA containing UV-induced photoproducts. However, unlike cells from the majority of XP patients, XP variant cells have a normal or nearly normal rate of nucleotide excision repair of such damage. To determine whether their IUV hypermutability reflected a slower rate of excision of photoproducts specifically during early S phase when the target gene for mutations, i.e., the hypoxanthine (guanine) phosphoribosyltransferase gene (HPRT), is replicated, we synchronized diploid populations of normal and XP variant fibroblasts, irradiated them in eariy S phase, and compared the rate of loss of cyclobutane pyrimidine dimers and 6-4 pyrimidine-pyrimidones from DNA during S phase. There was no difference. Both removed 94% of the 6-4 pyrimidine-pyrimidones within 8 h and 40%o of the dimers within 11 h. There was also no difference between the two cell lines in the rate of repair during G1 phase. To determine whether the hypermutability resulted from abnormal error-prone replication of DNA containing photoproducts, we determined the spectra of mutations induced in the coding region of the HPRT gene of XP variant cells irradiated in eariy S and G1 phases and compared them with those found in normal cells. The majority of the mutations in both types of cells were base substitutions, but the two types of cells differed significantly from each other in the kinds of substitutions observed either in mutants from S phase (P < 0.01) or from G, phase (P = 0.03). In the variant cells, the substitutions were mainly transversions (58% in S, 73% in G1). In normal cells, transversions were much rarer (8% in S, 24% in G1; P < 0.001 for S, P < 0.01 for G1). In the normal cells irradiated in S, the majority of the substitutions were G.C-*A.T, and most involved CC photoproducts in the transcribed strand. In the variant cells irradiated in S, substitutions involving cytosine in the transcribed strand were G.C--T.A transversions exclusively. G.C-oA.T transitions made up a much smaller fraction of the substitutions than in normal cells (P < 0.02), and all of them involved photoproducts located in the nontranscribed strand. The data strongly suggest that XP The hypermutability cannot be accounted for by errorprone excision repair because when synchronized populations of XP variant cells were irradiated at various times prior to S phase to allow different lengths of time for excision repair before DNA replication, the mutant frequency decreased as a function of time for repair until it reached background levels (32). One possible explanation for the UV hypermutability in the variant cells is that their replication fork encounters more photoproducts than does that of normal cells. This would be the case if the slightly lower rate of excision repair sometimes reported for XP variants reflects a significa...

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Section: Methodsmentioning

confidence: 99%

Evidence from mutation spectra that the UV hypermutability of xeroderma pigmentosum variant cells reflects abnormal, error-prone replication on a template containing photoproducts.

et al. 1993

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“…The supernatant was removed, and the cell pellet was resuspended in 5 ,ul of the cDNA cocktail described in detail by Yang et al (28). The reverse transcriptase reaction was performed at 37°C for 1 h to allow the cell membranes to be lysed by detergent and first-strand cDNA to be synthesized from total cytoplasmic poly(A) mRNA (28).…”

mentioning

confidence: 99%

“…The experimental conditions, optimized for preparing secondstrand HPRT cDNA, amplifying the cDNA 1011-fold by using two polymerase chain reaction stages of 30 cycles each, and sequencing the product directly by using three sequencing primers and a modified Sanger dideoxynucleotide procedure, have been described elsewhere (28).…”

mentioning

confidence: 99%

Cell cycle-dependent strand bias for UV-induced mutations in the transcribed strand of excision repair-proficient human fibroblasts but not in repair-deficient cells.

McGregor¹,

Chen²,

Лукаш³

et al. 1991

Mol. Cell. Biol.

140

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To study the effect of nucleotide excision repair on the spectrum of mutations induced in diploid human fibroblasts by UV light (wavelength, 254 nm), we synchronized repair-proficient cells and irradiated them when the HPRT gene was about to be replicated (early S phase) so that there would be no time for repair in that gene before replication, or in G, phase 6 h prior to S, and determined the kinds and location of mutations in that gene. As a control, we also compared the spectra of mutations induced in synchronized populations of xeroderma pigmentosum cells (XP12BE cells, which [HPRT]) is significantly higher than in cells irradiated in early G1 phase (9). This difference in frequency cannot be attributed to differences in the physical state of the DNA, since no such difference in mutation frequency is found when diploid xeroderma pigmentosum cells (XP12BE, complementation group A), which are virtually devoid of nucleotide excision repair (18), are irradiated in early S phase or in early G1 phase (9). These data suggest that S-phase replication is centrally involved in the conversion of potentially mutagenic DNA damage into mutations (fixation) and that excision repair prior to the onset of S phase decreases the frequency of mutants by eliminating such lesions.Of the two major classes of potentially mutagenic UV photoproducts, i.e., cyclobutane pyrimidine dimers (Py-Py dimers) and 6-4 pyrimidine-pyrimidone (6-4 Py-Py) lesions, the latter have been shown to be removed from human genomic DNA more rapidly than the former (15). It has also been shown that Py-Py dimers are excised more rapidly from an actively transcribed gene of human cells, the dihydrofolate reductase gene (DHFR), than from bulk DNA (1, 12). Moreover, Mellon et al. (14) demonstrated that there is a strand bias in the rate of removal of Py-Py dimers from the * Corresponding author. DHFR gene in human cells. The majority of such lesions are removed within 24 h, but dimers in the transcribed strand are excised much faster than those in the nontranscribed strand. Information on strand-specific excision repair of UV-induced damage from the HPRT gene of human cells is not yet available, but if such repair occurs, HPRT mutations induced by UV in excision repair-proficient human cells irradiated at the onset of S phase should arise from 6-4 Py-Py lesions and Py-Py dimers located in either strand of DNA. In contrast, the lesions responsible for the mutations induced in such cells allowed 6 h for repair before the onset of S phase should arise mainly from Py-Py dimers, and these should be located primarily in the nontranscribed strand. This switch in strands should not occur in XP12BE cells.There are no published data on the kinds and locations (i.e., the spectrum) of UV-induced mutations in an endogenous gene of human cells, nor are there data showing whether excision repair in such cells alters the spectrum. This study was designed to examine the spectrum of mutations induced by UV in the HPRT gene of diploid human cells and to determine, at the DNA ...

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“…The final product was run on a 1% agarose gel, stained with etidium bromide and observed under ultraviolet light. For direct sequencing of HPRT PCR products, the nested PCR products were purified by a QIAquick PCR Purification Kit (Qiagen GimbH) and aliquots of these PCR products were then sequenced by the Dana-Farber/Harvard Cancer Center DNA Resource Core (Boston, MA, USA) using three primers with the following sequences: Bases 264-252; 5'-ATT TCT ATT CAGT-3', bases 169-181; 5'-ATG GGA GGC CATC-3' and bases 405-417; 5'-TAT AAT TGA CACT-3' (Integrated DNA Technologies, Coralville, IA, USA), using an adapted method (25).…”

Section: Total Rna Isolation Rt-pcr For Determining Point Mutations mentioning

confidence: 99%

Intracellular and extracellular factors influencing the genotoxicity of nitric oxide and reactive oxygen species

Kim

2017

Oncology Letters

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Abstract. A number of factors affect cellular responses to nitric oxide (NO • ) and reactive oxygen species (ROS), including their source, concentration, cumulative dose, target gene and biological milieu. This limits the extrapolation of data to in vivo pathological states in which NO• and ROS may be important. The present study investigated lethality and mutagenesis in the HPRT and TK1 genes of human lymphoblastoid TK6 cells exposed to NO• and ROS derived from two delivery methods: A reactor system and a Transwell™ co-culture. The delivery of NO• into the medium at controlled steady-state concentrations (given in µM/min) and the production of NO• and ROS by activated macrophages, resulted in a time-dependent decrease in total cell numbers, and an increase in mutation frequency (MF), compared with untreated controls. This increase in MF was effectively suppressed by N-methyl-L-arginine monoacetate. Single base substitutions were the most common type of spontaneous and NO• induced mutations in HPRT, followed by exon exclusions and small deletions in both delivery systems. Among the single base pair substitutions, an equal frequency of four types of single base substitutions were identified in TK6 cells exposed to NO• delivered by the reactor system, whereas G:C to T:A transversions and A:T to G:C transitions were more frequent in the co-culture system. Taken together, these results demonstrate that both the delivery method of NO • and ROS, and the target genes are determinants of observed cytotoxic and mutagenic responses, indicating that these parameters need to be considered in assessing the potential effects of NO• and ROS in vivo.

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Amplification and direct nucleotide sequencing of cDNA from the lysate of low numbers of diploid human cells

Cited by 129 publications

References 14 publications

Evidence from mutation spectra that the UV hypermutability of xeroderma pigmentosum variant cells reflects abnormal, error-prone replication on a template containing photoproducts.

Evidence from mutation spectra that the UV hypermutability of xeroderma pigmentosum variant cells reflects abnormal, error-prone replication on a template containing photoproducts.

Cell cycle-dependent strand bias for UV-induced mutations in the transcribed strand of excision repair-proficient human fibroblasts but not in repair-deficient cells.

Intracellular and extracellular factors influencing the genotoxicity of nitric oxide and reactive oxygen species

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