Quantification of vitellogenin mRNA induction in mosquitofish (Gambusia affinis) by reverse transcription real-time polymerase chain reaction (RT-PCR)

A method to quantify induction of vitellogenin (Vtg) mRNA in adult male mosquitofish was developed. Male mosquitofish were exposed to 0, 1, 20 and 250 ng l^-1 17β-oestradiol (E₂) for 4 and 8 days in static exposures, and liver Vtg mRNA and 18S rRNA expression were quantified in duplex RT-PCR. Liver 18S rRNA expression was very consistent among individuals, and there was a highly significant increase in Vtg mRNA expression after exposure of mosquitofish for just 4 days at 250 ng l^-1 E₂. Lower doses did not induce Vtg mRNA expression even at 4 or 8 days. This method could be used as a rapid test to detect exposure of mosquitofish to oestrogenic chemicals. Further work is needed to determine if increased Vtg mRNA levels in male mosquitofish induce Vtg synthesis, and to determine the usefulness of the method in field sampling.     

Quantification of vitellogenin mRNA induction in mosquitofish (Gambusia affinis) by reverse transcription real-time polymerase chain reaction (RT-PCR)

Abstract

A method to quantify induction of vitellogenin (Vtg) mRNA in adult male mosquitofish was developed. Male mosquitofish were exposed to 0, 1, 20 and 250 ng l^?1 17β-oestradiol (E₂) for 4 and 8 days in static exposures, and liver Vtg mRNA and 18S rRNA expression were quantified in duplex RT-PCR. Liver 18S rRNA expression was very consistent among individuals, and there was a highly significant increase in Vtg mRNA expression after exposure of mosquitofish for just 4 days at 250 ng l^?1 E₂. Lower doses did not induce Vtg mRNA expression even at 4 or 8 days. This method could be used as a rapid test to detect exposure of mosquitofish to oestrogenic chemicals. Further work is needed to determine if increased Vtg mRNA levels in male mosquitofish induce Vtg synthesis, and to determine the usefulness of the method in field sampling.     

Amplification of a GC-rich sequence from barley by a two-step polymerase chain reaction in glycerol

PCR amplification of GC-rich sequences may fail due to poor denaturation or secondary structures that block elongation. Successful amplification of a 672-bp sequence encoding the barley α-amylase/subtilisin inhibitor (69% GC) was achieved in a simple two-step procedure with the addition of 20% glycerol and a high annealing temperature. This protocol may be useful for the amplification of other GC-rich sequences.     

Analysis of rev gene function on human immunodeficiency virus type 1 replication in lymphoid cells by using a quantitative polymerase chain reaction method.

Most detailed analyses of the human immunodeficiency virus type 1 (HIV-1) rev gene product have relied on transfection of subgenomic env constructs into cells in which amplification of the transfected DNA occurs. This was necessitated by difficulties in quantitating low-abundance HIV-1 mRNA species and in distinguishing different RNAs of similar sizes. We have modified the conventional polymerase chain reaction method for general use as an extremely sensitive procedure for quantitative analysis of RNA species. Using this method, we assessed the role of the HIV-1 rev gene in viral replication following mutagenesis of an infectious molecular clone, HIV-1JR-CSF. Following transfection of wild-type and mutant proviral constructs, we can specifically detect unspliced RNA and distinguish between the spliced tat-rev and nef mRNAs, which are not resolved by standard RNA analyses. Our results show that the rev protein of HIV-1JR-CSF simultaneously down regulates the expression of tat-rev and nef RNAs and up regulates the level of unspliced full-length HIV-1 RNA. A cis-acting element(s), located exclusively within the env sequences, is essential to exhibit this regulation. Fractionation of cells shows that the ultimate effect of Rev is to direct the appearance of unspliced or singly spliced RNAs in the cytoplasm. Models are discussed for possible mechanisms of Rev action.     

Analysis of DNA damage and repair in murine leukemia L1210 cells using a quantitative polymerase chain reaction assay.

The polymerase chain reaction (PCR) represents an alternative to the current methods for investigating DNA damage and repair in specific genomic segments. In theory, any DNA lesion which blocks Taq polymerase can be measured by this assay. We used quantitative PCR (QPCR) to determine the lesion frequencies produced by cisplatin and ultraviolet light (UV) in a 2.3 kilobase (kb) segment of mitochondrial DNA and a 2.6 kb segment of the DHFR gene in mouse leukemia L1210 cells. The frequency of UV-induced lesions increased linearly with dose, and was 0.58 lesions/10 kb/10 J/m2 in the mitochondrial DNA, and 0.37 lesions/10 kb/10 J/m2 in the DHFR gene. With cisplatin, the lesion frequency also increased linearly with dose, and was 0.17 lesions/10 kb/10 microM in the DHFR gene, and 0.07 lesions/10 kb/10 microM in mitochondrial DNA. This result is contrary to that of Murata et al., 1990 (1), in which mitochondrial DNA received greater cisplatin damage than did nuclear DNA. Using PCR to measure the repair of UV-induced lesions in the DHFR gene segment, we observed that less than 10% of the lesions were removed by 4 h, but over 70% of the lesions were removed by 8 h. Repair of 43% of UV-induced lesions in mitochondrial DNA was also observed during a 24 h period.     

Estrogen induces insulin-like growth factor-1 mRNA expression in the immortalized hippocampal cell: determination by quantitative real-time polymerase chain reaction

The basic mechanisms of nerve protection by estrogen remain to be clarified. This study was undertaken to confirm estrogen-induced insulin-like growth factor 1 (IGF-1) mRNA expression in the immortalized rat hippocampal cell H19-7 using a real-time quantitative polymerase chain reaction (PCR) assay, which has considerably increased accuracy and rapidity over other current methods. Upon stimulation by estradiol, the copy number of ER mRNA showed a 1.4-fold increase, and that of IGF-1 mRNA showed a 38.5-fold increase when compared with the control value. ICI182,780 inhibited the estradiol-induced upregulation of ER RNA completely, while it inhibited estradiol-stimulated IGF-1 mRNA expression partially. The increase of the copy number of IGF-1 mRNA was accomanied by enhancement of IGF-1 protein as observed by Western blot analysis.     

Cytokine response and polymerase chain reaction study of peripheral blood mononuclear cells in infants with human cytomegalovirus infection

Abstract We tried to detect human cytomegalovirus (HCMV) DNA in CD4 + and CD8 + T lymphocytes from fourteen infants with HCMV hepatitis using polymerase chain reaction (PCR) assay. HCMV was isolated from their urine and anti-HCMV IgM antibody was detected in their sera. One set of primers were designed from a region — a major immediate early (IE) gene. We detected HCMV IE DNA in the specimens obtained from six infants. HCMV IE DNA was detected from CD4 + cells in two cases and from CD8 + cells in one. In three cases, HCMV IE DNA was detected from both CD4 + and CD8 + cells. We also studied the relationship between HCMV infection and serum levels of cytokines. We determined serum levels of interleukin-4 (IL-4), tumor necrosis factor alpha (TNF-α) and soluble interleukin 2 receptor (sIL-2R) which were associated with the activation of T lymphocytes by enzyme immunoassay. In the acute phase of HCMV infection, titers of sIL-2R were correlated with serum levels of liver enzymes in some cases. IL-4 and TNF-α activities were not detected in sera. It is likely that expression of viral genome on T lymphocytes as well as activities of some cytokines are associated with active HCMV infection.     

Measuring growth of a phenanthrene-degrading bacterial inoculum in soil with a quantitative competitive polymerase chain reaction method

We measured growth of a phenanthrene-degrading bacterium, Arthrobacter, strain RP17, in Forbes soil, amended with 500 μg g(-1) phenanthrene using a quantitative competitive polymerase chain reaction method. The inoculum, which was not indigenous to Forbes soil, grew from 5.55x10(5) colony forming units (cfu) g(-1) to 1.97x10(7) cfu g(-1) within 100 h after the cells were added to the soil. Maximum population density was reached before the highest degradation rate was observed 150 h after the cells were added to soil. Population density remained stable even after 56% of the phenanthrene had mineralized. This study is one of the few documented examples of growth by a non-indigenous bacterium in a non-sterile soil amended with a pollutant.