细胞功能测定

Over the recent years, a large number of embryological studies with the zebrafish have provided substantial evidence of its usefulness for the investigation of the genetic and cellular basis of vertebrate development. With regard to the Hedgehog (Hh) pathway, forward as well as reverse gen ...

Xenopus embryos provide a powerful model system to investigate the complex molecular mechanisms, which are controlled by or control the activity of the Hedgehog (Hh) signaling pathway. The use of synthetic mRNA or antisense oligonucleotide (morpholino) microinjection into blast ...

Here, we describe methods for applying Sonic hedgehog (Shh) to developing chick limbs. The Sonic hedgehog gene is expressed in the polarizing region, a signaling region at the posterior margin of the limb bud and application of Shh-expressing cells or Shh protein to early limb buds mimics polari ...

The purification of recombinant versions of the N-terminal signaling fragment of Sonic hedgehog (ShhN) from E. coli, Hi-5™ insect cells, yeast, and mammalian cell sources reveals diverse post-translational modifications that affect the potency of the purified protein. Modificati ...

The Hedgehog (Hh)-signaling pathway has been intensively studied in the past decade. Increasing evidence suggests that dynamic formation of protein complexes plays a critical role in the organization and regulation of Hh signaling. Immunoprecipitation (IP) is a powerful tool to stu ...

Sequence analysis and comparative genomics are powerful tools to gain knowledge on multiple aspects of gene and protein regulation and function. These have been widely used to understand the evolutionary history and the biochemistry of Hedgehog (Hh) proteins, and the molecular contr ...

This chapter describes how to perform basic biochemical fractionations of Drosophila cells, and how to begin to characterize the proteins in the resulting fractions. The protocols include maintenance and transfection of Drosophila cell lines (Section 3.1.), hypotonic lysis (Sect ...

The GAL4/upstream activating sequence (UAS) system is one of the most powerful tools for targeted gene expression. It is based on the properties of the yeast GAL4 transcription factor which activates transcription of its target genes by binding to UAS cis-regulatory sites. In Drosophila, the ...

To fully understand how animals develop, it is often necessary to remove the function of a particular gene in a specific cell type or subset of cells. In Drosophila melanogaster, mosaic animals have been widely utilized to study cell fate, growth and patterning, and restriction of cell fate. This cha ...

Many of the genes of Drosophila melanogaster have their transcripts deposited in developing oocytes. These maternally loaded gene products enable an otherwise homozygous mutant embryo to survive beyond the first stage of development for which the gene product is required. Zygotic mu ...

RNA interference (RNAi) has become an irreplaceable tool for reverse genetics in plants and animals. The universality and specificity of this phenomenon allows silencing of virtually any chosen gene to examine its involvement in biological processes. Many strategies exist to reduce ...

Proper formation of different types of ribonucleoprotein (RNP)-RNA complexes is critical for the correct expression of genetic information at the posttranscriptional level. In the nucleus, RNA polymerase II transcripts bind to an abundant class of nuclear pre-mRNA/mRNA-bindi ...

The spliceosome is the catalytic entity that removes the introns from the primary transcripts in eukaryotes. The spliceosome consists of four small nuclear ribonucleoproteins (snRNPs), named U1,U2, U4/U6, and U5 snRNP, and numerous non-snRNP proteins. Each snRNP consists of one (U1, U2, and ...

Antisense oligonucleotides made of 2′-O-alkyl RNA are useful reagents for the study of the structure and function of ribonucleoprotein (RNP) particles. The nuclease resistant properties of these oligonucleotides, coupled with their ability to form specific and stable hybrids with ...

Within the last few years, it has become well established that a given nucleic acid binding protein has the potential to interact specifically with more than one target nucleic acid sequence. Various immunoprecipitation techniques have been developed to isolate specific DNA-protein ...

The problems of isolating sufficient quantities of rare RNAs for detailed biochemical analysis can be circumvented by synthesis of the desired RNA in vitro (1–3). Early methods of in vitro transcription included the use of eukary-otic cell extracts or Escherichia coli RNA polymerase to tra ...

To date, the most common approach to screening an expression library for an RNA-binding protein is one based on a procedure that was originally developed for cloning DNA-binding proteins (1,2): cDNA-encoded proteins from λ plaques are immobilized on nitrocellulose or nylon filters, which a ...

In the cell, all RNAs are involved in interactions with proteins that influence many aspects of their processing, localization, and expression (reviewed in refs. 1 and 2). In order to gain mechanistic insights into the role of proteins in these processes, one must (1) define the sequence elements in ...

RNA-binding proteins are involved in a variety of regulatory and developmental processes such as RNA processing, transport, and translation and are integral components of ribosomes, spliceosomes, nucleoli, and other ribonucle-oprotein particles. Proteins have been shown to in ...

RNA-binding proteins (RNA-BPs) play an essential role in key processes in all living organisms, from viruses to mammals. They are involved in RNA packaging, pre-mRNA splicing, translation, and RNA localization, to name a few. For these proteins to function properly it is imperative that they re ...