Background: Nuclear DNA content in plants is commonly estimated using flow cytometry (FCM). Plant material suitable for FCM measurement should contain the majority of its cells arrested in the G 0 /G 1 phase of the cell cycle. Usually young, rapidly growing leaves are used for analysis. However, in some cases seeds would be more convenient because they can be easily transported and analyzed without the delays and additional costs required to raise seedlings. Using seeds would be particularly suitable for species that contain leaf cytosol compounds affecting fluorochrome accessibility to the DNA. Therefore, the usefulness of seeds or their specific tissues for FCM genome size estimation was investigated, and the results are presented here. Methods: The genome size of six plant species was determined by FCM using intercalating fluorochrome propidium iodide for staining isolated nuclei. Young leaves and different seed tissues were used as experimental material. Pisum sativum cv. Set (2C ϭ 9.11 pg) was used as an internal standard. For isolation of nuclei from species containing compounds that interfere with propidium iodide intercalation and/or fluorescence, buffers were used supplemented with reductants.
The relationship between expression of a negative regulator of GA signal transduction (RGL2) belonging to the DELLA gene family and repression of Arabidopsis seed germination has been studied (Lee S, Cheng H, King KE, Wang W, He Y, Hussain A, Lo J, Harberd NP, Peng J [2002] Genes and Development 16: 646-658). There is one DELLA gene (LeGAI) present in tomato (Lycopersicon esculentum Mill.), which is expressed in both vegetative and reproductive tissues. During germination of wildtype tomato seed, there was no decline in the expression of LeGAI in either the embryo or the endosperm. Rather, LeGAI transcripts increased in these tissues following imbibition and remained high during and following germination. A similar increase in LeGAI transcripts occurred in the endosperm and embryo of GA-treated gib-1 mutant seed during and following germination. Likewise in soybean (Glycine max) seed, there was no decline in the expression of two DELLA genes in the radicle before or after germination. Upon reexamination of RGL2 in Arabidopsis seeds, a decline in its expression was noted but only after radicle emergence, i.e. after germination had been completed. Taken together, these data are consistent with GA-induced down-regulation of DELLA genes not being a prerequisite for germination of tomato, soybean, and Arabidopsis seeds.
The overrepresentation of phenotypically slow acetylators among patients with atopic allergy has been reported in previous studies. The N-acetyltransferase coding gene has not yet been investigated in allergic diseases. This study was designed to determine the differences in the distribution of mutation frequency and genotypes that encode normal and defective activity of N-acetyltransferase in children with atopic allergies compared with healthy children. In 56 children with documented inhalational, food, or mixed allergies and in 100 healthy control children with no clinical or laboratory signs of allergy, the genotype coding for N-acetyltransferase was identified by means of the polymerase chain reaction followed by analysis of restriction fragment length polymorphism. Nucleotide transitions in the following positions were investigated: 481 C-->T, 590 G-->A, 803 A-->G, and 857 G-->A, which enabled the identification of six genotypes, including the wild-type (wt) allele, and 16 genotypes, including mutated alleles (homozygotic and herterozygotic). The statistical analysis showed significant differences in the distribution of the frequency of the occurrence of mutated alleles and genotypes between the two groups of children. In 51 children (91%) with allergy, genotypes that encode acetylation defect were found; genotypes that code for normal N-acetyltransferase were observed in only five allergic children (9%). In the control group the distribution of genotypes coding for normal and defective N-acetyltransferase activity is 38% and 62%, respectively. Thus study enabled the conclusion that the slow acetylation genotype is a genetic marker of predisposition to atopy.
Loss of heterozygosity (LOH) in 16q appears in ~20-30% cases of Wilms' tumor. Within this region, known as common fragile site FRA16D, the WWOX tumor suppressor gene is located. Abnormalities of WWOX gene expression levels were observed in many tumor types and were associated with worse prognosis. The purpose of this study was to investigate the role of the WWOX tumor suppressor gene in Wilms' tumor samples. We evaluated the correlation between expression of WWOX and genes involved in proliferation (Ki67), apoptosis (BCL2, BAX), signal transduction (ERBB4, ERBB2, EGFR), cell cycle (CCNE1, CCND1), cell adhesion (CDH1) and transcription (TP73) using real-time RT-PCR in 23 tumor samples. We also analyzed the potential causes of WWOX gene expression reduction i.e., promoter methylation status (MethylScreen method) and loss of heterozygosity (LOH) status. We revealed a positive correlation between WWOX expression and BCL2, BCL2/BAX ratio, EGFR, ERBB4 isoform JM-a, TP73 and negative correlation with both cyclins. Loss of heterozygosity of the WWOX gene was observed only at intron 8, however, it had no influence on the reduction of its expression levels. Contrary to LOH, methylation of the region covering the 3' end of the promoter and part of exon 1 was associated with statistically significant reduction of WWOX gene expression levels. In the present study we reveal that in Wilms' tumors the WWOX expression levels are positively associated with the process of apoptosis, signal transduction through the ErbB4 pathway and EGFR and negatively with the regulation of the cell cycle (by cyclin E1 and D1). Moreover, our analysis indicates that in this type of tumor the expression of the WWOX gene can be regulated by an epigenetic mechanism--its promoter methylation.
The results show that the occurrence of adverse effects from co-trimoxazole is closely connected with the genotype coding for slow acetylation.
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