Identification of 3'-terminal exons from yeast artificial chromosomes.
نویسندگان
چکیده
We report an extension of 3'-terminal exon trapping technology to the identification of transcribed sequences from yeast artificial chromosomes (YACs). A 350-kb YAC containing mouse genomic DNA was gel-purified and used as the target DNA for the 3'-terminal exon trapping strategy. A novel direct ligation/transfection approach was employed to increase the efficiency of trapping 3'-terminal exons from recombinant vector-derived chimeric mRNA. The resulting RT-PCR product was then used to generate a plasmid library. Randomly chosen individual subclones from this library were sequenced, and the results indicate that 86% met sequence criteria characteristic of 3'-terminal exons, whereas 14% were background from identified sources. PCR mapping efforts suggest eight putative last exons present within this YAC, whereas RT-PCR studies demonstrate that three reside within valid expressed sequences.
منابع مشابه
USE OF VECTORETTE AND SUBVECTORETTE PCR FOR THE ISOLATION OF TERMINAL SEQUENCES FROM Y EAST ARTIFICIAL CHROMOSOME (YAC) CLONES
Development of yeast artificial chromosome (Y AC) vectors, molecular cloning of large segments of chromosomal DNA, and their propagation in yeast cells has become feasible. Overlapping Y AC provides a route to the development of physical maps of entire mammalian chromosomes. A rapid method was developed to isolate and sequence termini of Y AC inserts quickly. The Y AC clone is digested wit...
متن کاملTranscription and RNA processing of mammalian genes in Saccharomyces cerevisiae.
The recognition of mammalian genes encoded within a mouse yeast artificial chromosome (YAC) by the yeast transcription and RNA processing machinery was investigated. Transcripts from five genes known to be encoded by the YAC were all found in the total yeast RNA. Of 12 mouse introns assayed, six were correctly spliced by the yeast. However, an abnormal transcription of mouse DNA was also observ...
متن کاملTissue- and development-specific expression of multiple alternatively spliced transcripts of rat neuronal nitric oxide synthase.
Nitric oxide (NO) functions as an intercellular messenger and mediates numerous biological functions. Among the three isoforms of NO synthase that produce NO, the ubiquitously expressed neuronal NO synthase (nNOS) is responsible for a large part of NO production, yet its regulation is poorly understood. Recent reports of two alternative spliceforms of nNOS in the mouse and in man have raised th...
متن کاملHigh resolution restriction mapping of YACs using chromosome fragmentation.
The large insert size of yeast artificial chromosomes (YACs; 1) has made them ideal for the analysis of complex genomes. YACs can be restriction mapped with rare cutting enzymes but this does not provide the resolution required to investigate gene structure (such as locating exons and introns). Detailed mapping normally requires sub-cloning into cosmid or phage vectors. Here we describe a metho...
متن کاملEditorial Manager(tm) for Transgenic Research Title: a Transgenic Mouse Model Engineered to Investigate Human Brain-derived Neurotrophic Factor in Vivo
Brain-derived neurotrophic factor (BDNF) is an attractive component for the treatment of various neurodegenerative diseases such as Alzheimer's or Parkinson's disease. Innovative non invasive therapeutic approaches involve appropriate pharmacological induction of endogenous BDNF synthesis in brain. A transgenic mouse model has been established to study human BDNF gene expression and permit the ...
متن کاملذخیره در منابع من
با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید
عنوان ژورنال:
- PCR methods and applications
دوره 4 6 شماره
صفحات -
تاریخ انتشار 1995