Small RNAs, generally expressed at low levels, are difficult to reach usable levels from limited material. In this study, we have developed a novel method to amplify target RNA. The amplification procedure was carried out by sequential RT-PCR, effective separation, restriction enzymatic cleavage of cDNA strand, and run-off transcription in vitro of target RNA from its cDNA. Introduction of a unique stem-loop linker into cDNA strand is the key step to form a unique restriction enzyme recognition sequence that is not in cDNA sequence of target RNA. This method can be used to amplify RNA samples from various origins and has many advantages in amplifying unknown small RNAs and small RNA mixtures. The amplified RNA has the full sequence of original RNA except for an extra 5' G and an additional 3' A or C. The method worked well for amplifications of a microRNA, a piwi interacting RNA and two small RNA mixtures.

Motivation: The relationship between nucleosome positioning and gene regulation is fundamental yet complex. Previous studies on genomic nucleosome positions have revealed a correlation between nucleosome occupancy on promoters and gene expression levels. Many of these studies focused on individual nucleosomes, especially those proximal to transcription start sites. To study the collective effect of multiple nucleosomes on the gene expression, we developed a mathematical approach based on autocorrelation to relate genomic nucleosome organization to gene regulation.

Results: We found that nucleosome organization in gene promoters can be well described by autocorrelation transformation. Some promoters show obvious periods in their nucleosome organization, while others have no clear periodicity. The genes with periodic nucleosome organization in promoters tend to be lower expressed than the genes without periodic nucleosome organization. These suggest that regular organization of nucleosomes plays a critical role in gene regulation. To quantitatively associate nucleosome organization and gene expression, we predicted gene expression solely based on nucleosome status and found that nucleosome status accounts for ~25% of the observed gene expression variability. Furthermore, we explored the underlying forces that maintain the periodicity in nucleosome organization, namely intrinsic (i.e. DNA sequence) and extrinsic forces (i.e. chromatin remodeling factors). We found that the extrinsic factors play a critical role in maintaining the periodic nucleosome organization.

DNA damage induced by benzene reactive metabolites is thought of as an important mechanism underlying benzene hematotoxicity and genotoxicity, and genetic variation in cell-cycle control genes may contribute to susceptibility to chronic benzene poisoning (CBP). Using a case-control study that included 307 benzene-poisoned patients and 299 workers occupationally exposed to benzene in south China, we aimed to investigate the association between genetic polymorphisms of p53 and p21 and the odds of CBP. To investigate whether benzene exposure may influence mRNA expression of p53 and p21 in benzene-exposed workers, we also chose 39 CBP workers, 38 occupationally benzene-exposure workers, and 37 nonexposure workers in the same region of China. PCR-restriction fragment length polymorphism technique was applied to detect polymorphisms of p53 (rs17878362, rs1042522, and rs1625895) and p21 (rs1801270 and rs1059234), and real-time PCR was applied to detect the quantity of gene mRNA expression. We found that p21 C98A variant genotypes (CA+AA) or C70T variant genotypes (CT+TT) were associated with decreased odds of CBP [odds ratio (OR), 0.51; 95% confidence interval (95% CI), 0.32-0.83, and OR, 0.53; 95% CI, 0.29-0.95, respectively. Further analysis showed the decreased odds of CBP in the subjects with p21 CC/AT diplotype (OR, 0.51; 95% CI, 0.30-0.85). In addition, p53 mRNA expression of CBP workers or benzene-exposure workers was significantly lower than that of nonexposure workers. Although these results require confirmation and extension, our results show that polymorphisms in p21 may be protective against the risk of CBP in the Chinese occupational population. (Cancer Epidemiol Biomarkers Prev 2009;18(6):1821–8)

Drought is the most important environmental stress affecting agriculture worldwide. Exploiting yield potential and maintaining yield stability of crops in water-limited environments are urgent tasks that must be undertaken in order to guarantee food supply for the increasing world population. Tremendous efforts have been devoted to identifying key regulators in plant drought response through genetic, molecular, and biochemical studies using, in most cases, the model species Arabidopsis thaliana. However, only a small portion of these regulators have been explored as potential candidate genes for their application in the improvement of drought tolerance in crops. Based on biological functions, these genes can be classified into the following three categories: (1) stress-responsive transcriptional regulation (e.g. DREB1, AREB, NF-YB); (2) post-transcriptional RNA or protein modifications such as phosphorylation/dephosphorylation (e.g. SnRK2, ABI1) and farnesylation (e.g. ERA1); and (3) osomoprotectant metabolism or molecular chaperones (e.g. CspB). While continuing down the path to discovery of new target genes, serious efforts are also focused on fine-tuning the expression of the known candidate genes for stress tolerance in specific temporal and spatial patterns to avoid negative effects in plant growth and development. These efforts are starting to bear fruit by showing yield improvements in several crops under a variety of water-deprivation conditions. As most such evaluations have been performed under controlled growth environments, a gap still remains between early success in the laboratory and the application of these techniques to the elite cultivars of staple crops in the field. Nevertheless, significant progress has been made in the identification of signaling pathways and master regulators for drought tolerance. The knowledge acquired will facilitate the genetic engineering of single or multiple targets and quantitative trait loci in key crops to create commercial-grade cultivars with high-yielding potential under both optimal and suboptimal conditions.