Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-01-13
2001-10-30
Schwartzman, Robert A. (Department: 1635)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S325000, C435S375000, C536S023100, C514S04400A
Reexamination Certificate
active
06309830
ABSTRACT:
BACKGROUND OF THE INVENTION
The four nucleotide bases of DNA molecules carry genetic information. This information, in the form of codons of three contiguous bases is transcribed by mRNA and translated by tRNA and ribosomes to form proteins. The genetic code is the relation between a triplet codon and a particular amino acid. Of the sixty-four possible codon triplets which form the genetic code, there are three stop or terminating codons which are known to stop protein production at cellular ribosomes; the other sixty-one triplets in the code correspond to one or another amino acid. See Table 1
TABLE 1
UUU Phe
UCU Ser
UAU Tyr
UGU Cys
UUC Phe
UCC Ser
UAC Tyr
UGC Cys
UUA Leu
UCA Ser
UAA Stop
UGA Stop
UUG Leu
UCG Ser
UAG Stop
UGG Trp
CUU Leu
CCU Pro
CAU His
CGU Arg
CUC Leu
CCC Pro
CAC His
CGC Arg
CAU Leu
CCA Pro
CAA Gln
CGA Arg
CUG Leu
CCG Pro
CAG Gln
CGG Arg
AUU Lle
ACU Thr
AAU Asn
AGU Ser
AUC Lle
ACC Thr
AAC Asn
AGC Ser
AUA Lle
ACA Thr
AAA Lys
AGA Arg
AUG Met
ACG Thr
AAG Lys
AGG Arg
GUU Val
GCU Ala
GAU Asp
GGU Gly
GUC Val
GCC Ala
GAC Asp
GGC Gly
GUA Val
GCA Ala
GAA Glu
GGA Gly
GUG Val
GCG Ala
GAG Glu
GGG Gly
When genetic instructions are translated at ribosomes, the amino acids are strung together to form complex polypeptides. However, when a stop codon is read, it is interpreted as a stop signal terminating the protein production. The three stop codons are UAG (amber), UAA (ochre) and UGA (opal). Mutations that change a codon to stop codon are called nonsense mutations and, as a result, genetic phenotypes may not be expressed. Thus, despite the presence of a gene directing expression, a crucial protein may not be produced because an unwanted stop signal reaches a ribosome and terminates an unfinished protein.
Transfer RNAs (tRNAs) translate mRNA into a protein on the ribosome. Each transfer RNA contains an anti-codon region that hybridizes with mRNA, and an amino acid which may be attached to the growing peptide. The structural gene of tRNA is about 72-90 nucleotides long and folds into a cloverleaf structure. tRNAs are transcribed by RNA polymerase III and contain their own intragenic split promoters that become a part of the mature tRNA coding sequence (Sharp S. J., Schaack J., Coolen L., Burke D. J. and Soll D., “Structure and transcription of eukaryotic tRNA genes”,
Crit. Rev. Biochem,
19:107-144 (1985); Geiduschek E. O., and Tocchini-Valentini, “Transcription by RNA polymerase III,
Annu. Rev. Biochem.
57:873-914 (1988)).
Nonsense suppressors are alleles of tRNA genes that are altered in the anticodon so that they can insert an amino acid in response to a termination codon. For example, an ochre mutation results in the creation of a UAA codon in messenger RNA. An ochre suppressor gene produces tRNA with a AUU anticodon that inserts an amino acid at the UAA site permitting continued translation despite the presence of a nonsense codon.
A number of nonsense suppressor tRNA alleles have been identified in prokaryotes and eukaryotes such as yeast and C.elegans. However to date, no mammalian cell line containing functional suppressor tRNA has been isolated using classical genetic selection. Attempts to isolate suppressor tRNAs from higher eukaryotes resulted in the identification of an opal suppressor phosphoserine tRNA in the chicken genome (Hatfield D. L., Dudock B. S., and Eden F. C., “Characterization and nucleotide sequence of a chicken gene encoding an opal suppressor tRNA and its flanking DNA segments”,
Proc. Natl. Acad. Sci. U.S.A.,
80:4940-4944 (1983)), and later in the human genome (O'Neill V. A., Eden F. C., Pratt K., and Hatfield D. L., “A human opal suppressor tRNA gene and pseudogene”,
J. Biol. Chem.
260:2501-2508 (1985)). The two differ from each other at only a single nucleotide position. Suppressor tRNAs may also cause readthrough of the naturally occurring stop codons, thereby producing extended proteins with altered functions. Suppression of termination may be deleterious to the cell, although multiple natural stop codons at the end of the gene may provide safeguard from such harmful effects. The different suppressor tRNAs vary in their suppression efficiency. In
E.coli
and other systems the amber suppressors are relatively more efficient, ochre suppressors are less efficient while opal are the least, this suggests that the amber codons are used infrequently to terminate protein synthesis, while ochre and opal codons are more frequently used as natural termination signals.
Restoration of a normal phenotype by suppressors will depend on the type of amino acid inserted at the position of the nonsense codon. The inserted amino acid may be incompatible with the structure, function or stability of the gene product. Hence, there exists a need for a wide variety of suppressor tRNAs to insert different amino acids. Amber and ochre suppressors derived from a
Xenopus Laevis
tyrosine tRNA gene were shown to be functional in mammalian cells in transient transfection assays as well as in permanent cell lines (Laski F. A., Belagaje U. L., RajBhandary U. L. and Sharp P. A., “An amber suppressor tRNA gene derived by site-directed mutagenesis: cloning and expression in mammalian cells”,
Proc. Natl. Acad. Sci. USA,
79:5813-5817 (1982); Laski F. A., Belagaje R., Hudzoal R. M., Capecchi M. R., Palese P., RajBhandary U. L. and Sharp P. A., “Synthesis of an ochre suppressor tRNA gene and expression in mammalian cells”,
EMBO J
3:2445-2452 (1984); Hudziak R. M., Laski R. A., RajBhandary U., Sharp, P. A. and Capecchi M. R., “Establishment of mammalian cell lines containing multiple nonsense mutations and functional suppressor transfer RNA genes”, Cell 31:131-146 (1982)). Capone and co-workers similarly generated amber, ochre and opal suppressor tRNA genes derived from a human serine tRNA gene Capone J. P., Sharp P. A. and RajBhandary U. L., “Amber, ochre and opal suppressor tRNA genes derived from a human serine tRNA gene”,
EMBO J
4:213-221 (1985)).
In addition to permitting read-through of a mutation which causes a nonsense codon in the middle of a transcribed protein sequence, there are also times when one wants to manipulate a translation to truncate gene products. In either case, there exists a need for a suppression mechanism which would permit the cellular ribosomes to ‘read through’ such stop signals when they are unwanted. There is also a need for the opportunity to site specifically modify protein synthesis by deliberately altering the translation of the genetic code to learn about protein function.
It is an object of the present invention to provide novel nonsense suppressor tRNA's which are functional in cells and methods of use of the same in genetic engineering protocols.
SUMMARY OF THE INVENTION
According to the present invention novel oligonucleotide sequences which encode suppressor tRNAs or functional equivalents thereto are provided which, when introduced to cells containing a nonsense mutation, can suppress the expression of the nonsense stop codon allowing for complete translation of protein products. Based upon the knowledge of known human tRNA sequences, synthetic oligonucleotides relating to opal, amber, or ochre mutations are constructed which then may be used in any of a number of genetic engineering protocols.
Briefly, an oligonucleotide is synthesized which comprises the structural component of a known tRNA gene. The sequence of this oligonucleotide is designed based upon the known sequence with substitutions made in the anticodon region of the tRNA causing the specific tRNA to recognize a nonsense or any other specific or desired mutation. For example as shown in FIG.
2
and according to the invention, the sequence of human serine tRNA having an anticodon of TCG was modified to include a substitution of TCA the complement of the opal mutation to cause the tRNA to recognize the opal stop codon rather than the traditional serine codon.
Importantly the sequences for the oligonucleotides of the invention contain only the structural sequence encoding the tRNA molecule as well as a small portion (around
Link, Jr. Charles J.
Panchal Rekha G.
Epps Janet
Human Gene Therapy Research Institute
Schwartzman Robert A.
Zarley, McKee,Thomite, Voorhees & Sease
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