MicroRNAs: Genomics, Biogenesis, Mechanism, and Function

The human genome holds an extraordinary trove of information about human development, physiology, medicine and evolution. Here we report the results of an international collaboration to produce and make freely available a draft sequence of the human genome. We also present an initial analysis of the data, describing some of the insights that can be gleaned from the sequence.

/pdf/initial-sequencing-and-analysis-of-the-human-genome-5dapk1rsx1.pdf

Initial sequencing and analysis of the human genome.

抗原变异可使得多种致病微生物易于逃避宿主免疫应答。表达在感染红细胞表面的恶性疟原虫红细胞表面蛋白1（PfPMP1）与感染红细胞、内皮细胞、树突状细胞以及胎盘的单个或多个受体作用，在黏附及免疫逃避中起关键的作用。每个单倍体基因组var基因家族编码约60种成员，通过启动转录不同的var基因变异体为抗原变异提供了分子基础。

疟原虫var基因转换速率变化导致抗原变异[英]／Paul H, Robert P, Christodoulou Z, et al//Proc Natl Acad Sci U S A

http://bartellab.wi.mit.edu/publication_reprints/Bartel_Cell09.pdf

MicroRNAs: Target Recognition and Regulatory Functions

A procedure for extracting plasmid DNA from bacterial cells is described. The method is simple enough to permit the analysis by gel electrophoresis of 100 or more clones per day yet yields plasmid DNA which is pure enough to be digestible by restriction enzymes. The principle of the method is selective alkaline denaturation of high molecular weight chromosomal DNA while covalently closed circular DNA remains double-stranded. Adequate pH control is accomplished without using a pH meter. Upon neutralization, chromosomal DNA renatures to form an insoluble clot, leaving plasmid DNA in the supernatant. Large and small plasmid DNAs have been extracted by this method.

A rapid alkaline extraction procedure for screening recombinant plasmid DNA

A murine cell line (EN/NIH) containing the retroviral vector ZIPNeoSV(x)1 that was modified by deletion of the enhancer elements in the viral long terminal repeats has been used as an assay system to detect induced DNA rearrangements that result in activation of a transcriptionally silent reporter gene (neomycin phosphotransferase, neo ) encoded by the viral genome. The spontaneous frequency of G418 resistance is less than 10 -7 , whereas exposure to the tumor promoter 12- O -tetradecanoylphorbol-13-acetate (TPA) or the combination of UV irradiation plus TPA resulted in the emergence of drug resistant cell lines at a frequency of 5 per 10 6 and 67 per 10 6 cells, respectively. In several of the cell lines that were analyzed a low level of amplification of one of the two parental retroviral integrants was observed, whereas in others no alteration in the region of the viral genome was detected. To determine the effect of the SV40 large T antigen on induced DNA rearrangements, EN/NIH cells were transfected with a temperature sensitive ( ts ) mutant of SV40 T. Transfectants were maintained at the permissive temperature (33°C) for varying periods of time (1–5 days) in order to vary SV40 T antigen exposure, after which they were shifted to 39.5°C for selection in G418. The frequency of emergence of drug resistant cell clones increased with duration of exposure to large T antigen (9–52 per 10 6 cells over 1–5 days, respectively), and all cell lines analyzed demonstrated DNA rearrangements in the region of the neo gene. A novel 18-kilobase pair Xba I fragment was cloned from one cell line which revealed the presence of a 2.0-kilobase pair Eco RI segment containing an inverted duplication which hybridized to neo sequences. It is likely that the observed rearrangement was initiated by the specific binding of large T antigen to the SV40 origin of replication encoded within the viral genome. The investigations with phorbol esters, UV light, and the SV40 large T antigen demonstrate the utility of the EN/NIH cell lines for the study of induced DNA rearrangements and support the future use of this system to investigate the mechanism by which varied stimuli or specific gene functions promote DNA rearrangements.

https://cancerres.aacrjournals.org/content/canres/49/23/6640.full.pdf

Gene Activation by Induced DNA Rearrangements

https://cancerres.aacrjournals.org/content/canres/49/8/2188.full.pdf

Gene regulation and oncogenes

The crisis in funding: a time for decisions.

Publisher Summary
This chapter focuses on three classes of nuclear DNA-dependent RNA polymerases that have been identified in eukaryotic cells. Synthesis of mature RNA molecules requires additional enzymes and factors other than those needed to bring about accurate transcriptional initiation. For analysis by hybridization and SI nuclease digestion, it is important first to remove the template DNA. Titrations both of DNA and of extract yield nonlinear responses. At a constant extract concentration, measuring runoff transcription as a function of DNA concentration yields a threshold DNA concentration below which no transcription occurs and an inhibitory effect of high DNA concentration. For a given promoter, short runoff transcripts have a higher optimum DNA concentration than longer runoff transcripts. A number of inhibitory activities can be removed by fractionation on phosphocellulose, yielding a more efficient transcription extract.

36 – In Vitro Transcription: Whole-Cell Extract

Berries are a rich source of polyphenolic compounds such as flavonoids, including anthocyanins, with wide-ranging potential therapeutic effects 1. It has been demonstrated previously that intestinal uptake of dietary glucose is impeded by the presence of polyphenolic compounds; however, little is known regarding the genes involved in this mechanism 2. The aim of this study was to investigate the effects of an anthocyanin-rich berry-extract on dietary glucose transporting genes in Caco-2 cells. Human intestinal Caco-2 cells were cultured for 19 d and were then treated for 16 h with an anthocyanin-rich berry-extract (OptiBerry; InterHealth Nutraceuticals, Benicia, CA, USA) at a final concentration of 0.5% (w/v). Subsequently mRNA was isolated and used for the quantitative real-time polymerase chain reaction (qRT–PCR), using 18S and GAPDH as housekeeping genes. Gene expression data are expressed as means ( SEM) relative expression ratios of control; n = 5/6. The expression of GLUT2 (apical/basolateral monosaccharide transporter), GLUT5 (apical fructose transporter) and SGLT1 (apical sodium/glucose co-transporter) was decreased by the berry-extract treatment. GLUT2, GLUT5 and SGLT1 expression as a ratio of the control was as follows: GLUT2 (0.20 0.02; P = 1.9 · 10 ), GLUT5 (0.57 0.08; P = 2.5 · 10 ) and SGLT1 (0.45 0.03; P = 3.1 · 10 ). GLUT2, GLUT5 and SGLT1 have previously been identified as therapeutic targets for dysregulated glucose metabolism. In this study, the expression of these genes in Caco-2 cells was decreased by treatment with an anthocyanin-rich berry-extract. Studies are in progress to investigate the biological relevance of the observed effects in relation to berry consumption and the absorption of dietary sugar.

/pdf/anthocyanin-rich-berry-extract-treatment-decreases-5eltvwu4tb.pdf

Phillip A. Sharp

Papers

Gene Activation by Induced DNA Rearrangements

Gene regulation and oncogenes

The crisis in funding: a time for decisions.

36 – In Vitro Transcription: Whole-Cell Extract

Anthocyanin-rich berry-extract treatment decreases expression of dietary glucose transporter genes in human intestinal Caco-2 cells