实验基础

The development of efficient methods for manipulation of cell-specific gene expression in vivo is of great value for gaining new insights into complex biological systems, and for advancing gene therapy for human diseases. Transcript-specific downregulation of RNA levels and activ ...

RNase P is an enzyme that cleaves tRNA precursors to generate the 5′ termini of mature tRNAs (1). Recently, the author developed a method for specific inactivation of any RNA using human RNase P (2). This method involves the design of a small RNA, known as external guide sequence (EGS). EGS RNA, when complexed w ...

Most human diseases are a result of the dysfunction of cellular genes or a result of the invasion of cells by foreign genetic materials. Blocking disease-causing genes from making the proteins they specify is a strategy for combating disease processes. Antisense and ribozyme are two widely kn ...

Inactivation of gene expression by antisense mechanisms in general and by ribozymes in particular show great promise as a research tool as well as therapeutic agent in selectively reducing mRNA species and thus decreasing subsequent protein synthesis (for reviews see refs. 1 and 2).

A number of different approaches may be taken to show the effects of ribozymes in cells, yet they all have shortcomings. One major difficulty is that the concentration of target RNAs in cells is often very low. This is confounded by the fact that the cleavage products are generally degraded very quickly ...

Hepatitis delta virus (HDV) is an infectious, subviral, pathogen that is associated with a high incidence of fulminant hepatitis in humans. It consists of a closed-circular, single-stranded, RNA genome of about 1700 nucleotides in length that is replicated by a rolling-circle mechanism ( ...

The minimum catalytic center of (-)s TRSV was identified, biochemically characterized and named the hairpin ribozyme (1, 2). Following initial identification of the minimum catalytic sequence, we identified a trans-catalytic reaction and biochemically characterized this rea ...

The development of acquired immunodeficiency syndrome (AIDS) generally takes of the order of ten years with no effective therapy currently available (1,2). HIV, the causative agent, exhibits tropism for CD4+ T-lymphocytes, the primary targets for HIV infection in vivo. Infection of these ...

Expression of DNA methyltransferases (MTases) in yeast, which has a naturally unmethylated genome, enables the study of chromatin structure in intact cells. Initial studies, employing in vivo expression of dam MTase, which catalyzes the production of N6-methyladenine (6meA) at GATC s ...

Short (153–350 bp) DNA fragments containing single nucleosome cores have been employed for investigation of a variety of topics, including binding of regulatory transcription factors to nucleosomes (1) and mechanism of transcription of nucleosomal templates (2). The major advant ...

It has become increasingly clear over the last decade that chromatin structure and gene regulation are intricately intertwined. Different regulatory states of a given gene are frequently accompanied by changes in nuclease hypersensitive sites and nucleosome positioning (1–4). C ...

Gene expression is regulated by complex mechanisms involving dynamic interactions between cis-acting elements and trans-acting factors in a highly structured chromatin environment. Investigations of protein/DNA interactions in vitro may not have relevance to a living cell s ...

General remodeling of chromatin is associated with events determining cell fate and the expression of specific genetic programs (1,2). In almost every case there is a tight link between these chromatin remodeling events and a drastic modification of the cell cycle parameters. One of the most s ...

Eucaryotes use a common theme for packaging genomic DNA: 146 bp of DNA are wrapped around an octameric protein core consisting of two molecules each of the histones H2A, H2B, H3, and H4. Although this nucleosomal arrangement is ubiquitous throughout the genome, different chromosomal portions ...

The method described here is based on a technique developed to analyze the chromatin structure of the SV40 origin of replication, and also alterations in nucleosomal structures in the hsp70 promoter of Drosophila after heat shock (1,2). It relies on the ability of formaldehyde to crosslink pro ...

Protein-nucleic acid complexes play a crucial role in the events involved in gene expression and regulation. Direct and powerful approaches in studying this regulation are footprinting and protein-DNA crosslinking. Protein-DNA crosslinking detects the presence of a protein on a g ...

The protein-DNA complexes that make up the chromosome serve not only to package the genomic DNA within the confines of the nucleus but to directly participate in the efficient and controlled utilization of the DNA for nuclear processes such as replication, transcription, recombination, a ...

In vivo UV crosslinking permits direct analysis of protein-DNA interactions in intact cells. This technique has been used to study DNA binding by a wide variety of proteins including RNA Polymerase II, Topoisomerase I, and sequence specific transcription factors such as Even-Skipped, Ze ...

The position of a nucleosome describes the arrangement of its core 147 base pairs of DNA relative to the core histone octamer (1). The DNA superhelix spiraling around the histone octamer has an outer, solvent exposed face and an inner, histone-associated face (2). Fixing of a particular exposed hel ...

Researchers often think of nucleosomes as inert and static structures. But the laws of physical chemistry—which apply to any molecular complex—remind us that this picture cannot be correct. Indeed, many early studies revealed that nucleosomes undergo a complex set of assembly/disass ...