Hamster EF-1 alpha transcriptional regulatory DNA

A transcriptional regulation, EF-1 technology, applied in recombinant DNA technology, DNA/RNA fragments, stable introduction of foreign DNA into chromosomes, etc.

Inactive Publication Date: 2009-11-04
ICOS CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The TATA box is the binding site for a ubiquitous transcription factor named TFIID, but as mentioned, the transcription of promoter sequences in most genes is mainly influenced by additional regulatory DNA sequences that enhance or repress gene transcription

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] CHEF1 clone

[0021] The Chinese Hamster Ovary Genomic DNA Bank (CHO-K1) (Stratagene, La Jolla, CA) was screened with the cDNA encoding human EF-1α in order to isolate the hamster homolog of the human EF-1α gene.

[0022] CHO-K1 library (Lambda FIX Partial digestion of Sau3A in II cloning vector) was obtained from Stratagene (La Jolla, CA) as host cell lines XL-1-Blue MRA and XL1-Blue MRA (P2). The host cells were prepared according to the manufacturer's suggested protocol with the following modifications.

[0023] Briefly, cells taken from glycerol stocks were streaked on LB plates without antibiotics. Single clones were screened for inoculation in liquid LB medium and cultured to late log phase and OD 600 About 0.9. At this point, cells were stored according to the manufacturer's recommendations or used immediately as described below.

[0024] When preparing plated cultures, single colonies were picked from plates prepared as above for inoculation and the cells ...

Embodiment 2

[0046] Secondary cloning of EF-1α regulatory sequences

[0047] To determine the size of the insert fragment EF-1α DNA, the phage DNA prepared as in Example 1 was digested with NotI, and the resulting restriction fragments were separated on 0.6% 1x TAE agarose gel. Clones 2.12 and 2.17 showed the same enzymatic banding pattern, with bands at 11 kb and 4.5 kb flanking the expected 19 kb and 10 kb lambda fragments. Clones 7.44 and 40.24 also showed the same digestion band pattern with 12 kb and 7 kb insert bands which together with the digestion data of clones 2.12 and 2.17 indicated the presence of an internal NotI restriction enzyme recognition in the EF-1α DNA point. Inserts derived from clones 2.12 and 7.44 were subcloned as follows.

[0048] Phage DNA (60 microliters) prepared as described in Example 1 was digested with NotI, after which 20 microliters of 3M sodium acetate and 400 microliters of 100% ethanol were added to precipitate the digested DNA. Precipitated DNA wa...

Embodiment 3

[0063] Characterization of CHEF regulatory DNA

[0064] The sequence upstream of the hamster EF-la gene and including the ATG start codon is given in SEQ ID NO:1. The majority of identifiable transcription factor binding sites that emerged were located 3' to the upstream SacI site, 5' to the initiation ATG codon of the EF-1[alpha] gene, 1.56 kb. However, expression vectors including the CHEF1 sequence described below also contained a 2 kb CHEF1 sequence 5' to the SacI site.

[0065] Sequencing revealed the presence of a fully consensus TATA box of approximately 1 kb 5' to the initiation ATG start codon, and the region between the upstream SacI site and the TATA box contained multiple potential transcription factor binding sites[ Boulikas, Crit.Rev.Euk.Gene Exp.4:117-321 (1994)], including Sp1 site (SEQ ID NOs: 16 or 17), ATF site (SEQ ID NO: 18), and NF-1 site (SEQ ID NO: 19).

[0066] GGCGGG SEQ ID NO: 16

[0067] GGGCGG SEQ ID NO: 17

[0068] TGA...

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PUM

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Abstract

The present invention relates to regulatory DNA sequences derived from the hamster EF-1 alpha gene. The invention further encompasses expression constructs comprising regulatory DNA of the invention, host cells transformed or transfected with the regulatory DNA and methods of increasing gene transcription utilizing the regulatory DNA. Expression constructs comprising regulatory DNA of the invention operably linked to specific gene sequences are also comprehended.

Description

[0001] This application is a divisional application of an invention patent application with an application date of May 1, 1998, an application number of 98800913.7, and an invention title of "Hamster EF-1α Transcription Regulation DNA". Background of the invention [0002] Transcription of any given eukaryotic gene occurs by one of three RNA polymerases, each of which acts on some specific subset of genes. If, say, the transcription of large ribosomal RNAs is performed by RNA polymerase I and the transcription of small ribosomal RNAs and tRNAs by RNA polymerase III, protein-coding DNA sequences and most small nuclear RNAs are transcribed by RNA polymerase II. For each type of gene, transcription requires the interaction of the polymerase with the gene promoter sequence and formation of a stable transcription initiation complex. In general, transcription of any of the three polymerases also requires some binding factor to interact with the promoter sequence and recognition of t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N15/63C12N15/67C12N15/85C12N15/90C12N5/10C12N1/15C12N1/19C12N1/21C07K14/47C12N15/09A01K67/027C12NC12N1/00C12N15/11C12N15/62
CPCC12N15/85C07K14/4702C07K2319/00C07K14/47A01K2217/05C12N15/67C12N15/63
Inventor D·S·阿里森
Owner ICOS CORP
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