遗传学实验技术

Mammalian gene expression can be regulated through various post-transcriptional events, including altered mRNA stability, translational control, and RNA-processing events such as 3′-end formation or polyadenylation (pA). It has become clear in recent years that pA is governed by ...

Most metazoan genes are alternatively spliced, and a large number of alternatively spliced isoforms are likely to be functionally significant and expressed at specific stages of pathogenesis or differentiation. Splicing changes usually only affect a small portion of a gene, and these ...

RNA localization is a cellular process to spatially restrict translation of specific proteins to defined regions within or between cells. Most localized mRNAs contain cis-acting localization elements in the 3′-untranslated region (UTR), which are sufficient for localization of an ...

All eukaryotic cells display a dramatic partitioning of mRNAs between the cytosol and endoplasmic reticulum (ER) compartments—mRNAs encoding secretory and integral membrane proteins are highly enriched on ER-bound ribosomes and mRNAs encoding cytoplasmic/nucleoplasm ...

Regulating gene expression at the translational level controls a wide variety of biological events such as development, long-term memory, stress response, transport and storage of certain nutrients, and viral infection. Protein synthesis at steady-state level can be directly mea ...

The number and distribution of ribosomes on a transcript provide useful information in ascertaining the efficiency of translation. Herein we describe a direct method to determine the association of ribosomes with specific regions of an mRNA. The method, termed Ribosome Density Mappi ...

The inability of structural elements within a reporter mRNA to impede processive decay by the major 5′ and 3′ exonucleases has been a major obstacle to understanding mechanisms of vertebrate mRNA decay. We present here a new approach to this problem focused on quantifying the decay of individual ...

Sequence elements within mRNA-untranslated regions and their binding partners are key controllers of mRNA stability. Changes in mRNA stability can often be detected by changes in steady-state mRNA abundance, or a more careful analysis of mRNA half-lives can be performed following tra ...

Cytoplasmic extracts have proven to be a versatile system for assaying the mechanisms and interactions of RNA metabolism. Using Aedes albopictus (C6/36) cells adapted to suspension culture, we have been able to faithfully reproduce and manipulate all aspects of mRNA decay in vitro. Descr ...

相关专题 一、生物分子有机溶剂沉淀分离的原理： 有机溶剂对许多溶于水的生物小分子以及核酸、多糖、蛋白质等生物大分子都能发生沉淀作用。有机溶剂主要是降低溶液的介电常数，从而增强分子之间的相互作用使其溶解度降低而析出。对具有表面水层的生物大分子，有机溶剂可破坏溶质分子表面的水膜，使这些大分子脱水而相互聚集析出。不同溶质要求不同浓度的有机溶剂，可用有机溶剂进行分步沉淀。最后经离心机分离后获得沉淀物和上清液。 二、生物分子有机溶剂 ...

相关专题 一、生物分子透析分离的原理： 天然或人工半透膜只允许小分子通过而阻碍大分子通过，当膜的两侧存在小分子浓度差时，小分子从高浓度一侧向低浓度一侧扩散直到平衡。通过离心机分离不断去除扩散出来的小分子，从而达到分离纯化的目的。 二、影响生物分子透析分离的因素： 1、半透膜的通透性：半透膜的通透性取决于膜孔径的大小，在能阻碍大分子扩散的前提下，孔径越大透析速度越快。 2、温度：温度越高透析速度越快。 3、压力：增大膜压力可使分离速度加快。

IMGT/V-QUEST is the highly customized and integrated online IMGT� tool for the standardized analysis of the immunoglobulin (IG) or antibody and T cell receptor (TR) rearranged nucleotide sequences. The analysis of these antigen receptors represents a crucial challenge for the study of t ...

DNA methylation of retrotransposons and imprinted genes is accurately regulated in spermatogenesis. In particular, CpG methylation of long interspersed elements-1 (LINE1 or L1) and intracisternal A-particle (IAP) retrotransposons during spermatogenesis has been well ch ...

Piwi-interacting RNAs (piRNA) are fully modified by 2′-O-methylation at their 3′-termini. This terminal methylation is required to prevent 3′-nucleotide addition, which serves as a tag for destabilization. In this chapter, we describe biochemical and mass spectrometric analyses of 2 ...

Piwi proteins and their small-RNA partners, piwi-interacting (pi)RNA, form a natural mechanism that prevents the deleterious activity of transposable elements in the germ line of metazoan species. The piRNA pathway relies on extended noncoding genomic regions, dubbed piRNA clust ...

Classical biochemical approaches have made great contribution to our current understanding of how small interfering RNA (siRNA) and microRNA (miRNA) are produced and function. However, it has been challenging to establish in vitro systems that can dissect the mechanism of PIWI-inte ...

Emerging evidence indicates that PIWI proteins, in collaboration with PIWI-interacting RNAs (piRNAs), play a critical role in retrotransposon silencing in Drosophila gonadal somatic and germ-line cells. The recent establishment of female germ-line stem cells/ovarian somat ...

The Drosophila piwi gene has multiple functions in soma and germ cells. An in vitro system provides a powerful tool for elucidating PIWI function in each cell type using stable cell lines originating from germline stem cells (GSCs) and ovarian soma of adult ovaries. We have described methods for the ...

The generation of high-resolution maps of the epigenome is crucial to research in epigenetics, developmental biology, and stem cell biology. In recent years, small RNA pathways have been implicated in epigenetic regulation. All small RNA pathways involve Argonaute proteins as their key ...

The small noncoding RNAs in plants are categorized into two major classes, 21-nucleotides (nt) micro RNA (miRNA) and 21- or 24-nt small-interfering RNA (siRNA). ARGONAUTE (AGO) proteins associate with small RNAs and play central roles in transcriptional and posttranscriptional gene re ...