Chemistry: molecular biology and microbiology – Vector – per se
Patent
1996-04-19
1998-03-03
Ketter, James
Chemistry: molecular biology and microbiology
Vector, per se
536 231, 536 241, C07H 2104
Patent
active
057233322
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to DNA useful as an enhancer of translation of nucleic acids in the production of proteins therefrom.
2. Description of the Related Art
Kinesins are molecular motor proteins implicated in the intracellular transport of organelles in brain cells and in the movement of chromosomes along microtubules during cell division. In sea urchin and mammalian cells, kinesins have been characterized as tetrameric proteins. Two subunits are the two heavy chains (.alpha. chains) of a relative molecular mass of approximately 120 kDa and two light chains (.beta. chains) of approximately 70 kDa. Intracellular organelles move along microtubules inside the cell by attaching to the tetrameric .alpha..alpha./.beta..beta. kinesin molecular motor. The .alpha. chains provide the tubulin binding site and the ATPase domain, whereas the .beta. chains are responsible for the specific attachment of the organelle to be moved by the kinesin tetramer.
At present three rat brain kinesin .beta. chain cDNAs have been cloned, (J. L. Cyr et al., Proc. Natl. Acad. Sci. USA 88, 10114-10118) and a partial cDNA sequence (named EST00761) of human brain origin has been entered on the EMBL database as a result of the human genome sequencing work reported by M. D. Adams et al., (Nature 355, 632-634 (1992)). The three rat cDNAs are the product of one gene spliced differentially at the 3'-end, producing different COOH terminal ends in the protein and thereby predicting three different isoforms of .beta.-light chain, designated "A", "B" and "C". This mechanism seems to confer the kinesin specificity for organelle binding.
A human .beta.-kinesin cDNA sequence of length 2309 bp has been entered on the EMBL database under Accession No. LO4733 by L. B. Lachman et al. and published as Cabeza-Arveliz et al., DNA Cell Biol. 12, 881-892, 1993.
SUMMARY OF THE INVENTION
It has now been found that the .beta.-chain kinesin genomic DNA contains a region of DNA of high secondary structure which provides a strong enhancer of translation (not to be confused with an enhancer of promotion). In the human kinesin, the secondary structure lies within the first exon of the gene, which provides the 5'-untranslated end of the mRNA. It takes the form of a double hairpin loop which is illustrated for the mRNA in FIG. 4 of the drawings and listed in the sequence listing as SEQ ID NO:6.
The invention includes isolated DNA from part of the .beta.-light chain gene of a kinesin as well as various constructs in which this DNA serves as a translational enhancer. Thus, in one aspect, it includes isolated DNA from the 5'-end of the allelic gene which expresses the .beta.-light chain of a kinesin, said DNA being a translational enhancer and comprising a region of high secondary structure, capable of being represented as a hairpin loop; or a translation-enhancing substitution or deletion mutant thereof.
The isolated DNA thus includes the secondary structure region and optionally additional DNA to the 5'- or 3'-end thereof, which may be the DNA native to the same kinesin gene. Thus, the isolated DNA can include any or all such DNA extending in the 5'-direction back to the 5'-end of the .beta.-light chain gene. Thus, it may include part or all of the promoter region which extends upstream of the mRNA start site. It may include part or all of the first intron (which lies between the first exon coding for untranslated mRNA and the second exon coding for the N-terminal region of the .beta.-kinesin protein).
Since the .beta.-kinesin promoter is not particularly strong, the invention can be put to better use by linking the translational enhancer DNA to a strong eukaryotic promoter. Thus, in another aspect the invention provides a construct for enhanced expression of non-.beta.-kinesin DNA encoding a protein, comprising (1) a eukaryotic promoter, which is preferably a foreign promoter stronger than that of the native .beta.-kinesin gene, (2) downstream thereof, translational enhancer DNA of the invention and (3), downstr
REFERENCES:
patent: 4937190 (1990-06-01), Palmenberg et al.
J.L. Cyr et al. "Molecular genetics of kinesin light chains: Generation of isoform . . . " Proceedings of the Natl Acad. Of Sci. Of the USA, Nov. 15, 1991, vol. 88 No. 22, pp. 10114-10118.
Wedaman, K., et al., J. Mol. Biol., vol.231, pp. 155-158, 1993.
Invirtogen Catalog, especially pp. 24, 27, and 29, 1991.
British Technology Group Limited
Ketter James
Yucel Irem
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