Codon pair utilization

• US 5,082,767 A
• Filed: 02/27/1989
• Issued: 01/21/1992
• Est. Priority Date: 02/27/1989
• Status: Expired due to Term

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Hatfield, G. Wesley, Gutman, George A.
• Primary Examiner(s): Wax, Robert A.
• Assistant Examiner(s): Marschel, Ardin H.
• Application Number:
  US07/316,321
• Time in Patent Office: 1,058 Days
• Field of Search: 435/6, 435/91, 435/69.1, 435/172.3, 436/501, 536/26, 536/27, 536/28, 935/17, 935/78, 935/88
• US Class Current: 435/6.14
• CPC Class Codes: C07K 1/1136 by reversible modification of the secondary, tertiary or quarternary structure, e.g. using denaturating or stabilising agents
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6888 for detection or identification of organisms
• CPC Class Codes: C12Q 2600/158 Expression markers

Abstract

A method for determining the pattern of nonrandom codon pair usage of an organism, comprising the steps of obtaining nucleotide sequence data for the organism, determining from the data the number of codons represented in at least a portion of the sequence and the frequency of usage of at least some codons in the portion, determining from the frequency the expected number of occurrences of at least some codon pairs, if they are paired in a random manner, and comparing the expected number with the actual number of occurrences to determine relative codon pairing preferences. The codon pairings of organisms are highly nonrandom, and differ from organism to organism. This information is used to construct and express altered or synthetic genes having desired levels of translational efficiency, to determine which regions in a genome are protein coding regions, to introduce translational pause sites into heterologous genes, and to ascertain relationship or ancestral origin of nucleotide sequences.

Claims

1. A method for determining relative native codon pairing preferences in an organism, comprising the steps of:

obtaining nucleotide sequence data for said organism;

determining from said data the number of codons represented in at least a portion of said sequence and the frequency of usage of at least some individual codons in said portion;

determining from said individual codon frequencies the expected number of occurrences of at least some codon pairs, if said codons are paired in a radon manner;

comparing the expected number with the actual number of occurrences to determine said relative condon pairing preferences;

determining preferred amino acid pairings of said organism and eliminating any amino acid pair bias prior to determining said codon pairing preferences; and

altering a gene for expression in said organism by substituting at least one codon for an existing codon in said gene to alter codon pairing in accordance with said codon pairing preferences to change the translational kinetics of said gene in a predetermined manner.

2. The method of claim 1, in which said codon pairing is altered to increase the translational kinetics of at least a portion of said gene.

3. The method of claim 2, in which said codon pairing is altered to increase the number of codon pairs that, in comparison to random codon pair usage, are the more abundant and yet more under-represented codon pairs in said organism.

4. The method of claim 1, in which said codon pairing is altered to decrease the translational kinetics of at least a portion of said gene.

5. The method of claim 4, in which said codon pairing is altered to increase the number of codon pairs that, in comparison to random codon pair usage, are the less abundant and yet more over-represented codon pairs in said organism.

6. The method of claim 1, wherein at least 100% of the existing condons pairs are changed.

7. The method of claim 1, further comprising the step of constructing a synthetic gene coding for a predetermined polypeptide for expression in said organism, and selecting codons for amino acids of said polypeptide in accordance with the relative native condon pairing preferences of said organism to achieve a desired level of translational kinetics of said gene.

8. The method of claim 7, wherein at least 10% of said codon pairs are selected in accordance with the relative native codon pairing preferences of said organism.

9. The method of claim 7, in which said codon pairing is selected to increase the number of codon pairs that, in comparison to random codon pair usage, are underepresented codon pairs in said organism.

10. The method of claim 7, in which said codon pairing is selected to increase the number of codon pairs that, in comparison to random codon pair usage, are over-represented codon pairs in said organism.

21. A method for expressing a gene in an organism, said gene having altered translational kinetics, comprising the steps of:

introducing a gene prepared according to the method of claim 1 into said organism; and

expressing said gene in said organism.

22. A method for expressing a gene in an organism, said gene having altered translational kinetics, comprising the steps of:

introducing a gene prepared according to the method of claim 7 into said organism; and

expressing said gene in said organism.

11. A method for making at least a portion of a synthetic gene coding for a polypeptide, wherein said gene is intended for expression in a particular organism, comprising the steps of:

obtaining information regarding the relationship between codon pairing and translational kinetics for said organism; and

for at least one amino acid pair in said polypeptide, selecting from among possible codon pairs a pair that provides desired translational kinetics; and

providing said selected codon pair in said gene in place of a less-preferred pair.

12. The method of claim 11, wherein at least three codon pairs are selected and provided in accordance with said information.

13. The method of claim 11, wherein said selected codon pair is preferred by said organism in genes expressed at low levels of less than about 1000 copies per cell.

14. The method of claim 11, wherein said selected codon pair is preferred by said organism in genes expressed at intermediate levels of from about 1000 to about 10,000 copies per cell.

15. The method of claim 11, wherein said selected codon pair is preferred by said organism in genes expressed at high levels of greater than about 10,000 copies per cell.

16. The method of claim 11, wherein said selecting step affects at least 10% of the codons of said gene.

17. A method for altering a gene of a first organism for expression in a second organism, comprising the steps of:

obtaining information regarding native codon pairing preferences for at least some genes of said second organism; and

replacing one or more existing codon pairs of said gene of said first organism in accordance with said information with codon pairs coding for the same amino acids but for which said second organism has different preferences that it does for said existing codon pairs, to alter the translational kinetics of said pairs in a predetermined manner.

18. The method of claim 17, wherein said replacing step alters at least one codon pair to a pair over-represented or under-represented in said genes of said second organism to the same general degree as was said replaced pair in said first organism.

19. The method of claim 17, wherein said replacing step increases the translational speed of said codon pairs.

20. The method of claim 17, wherein said replacing step decreases the translational speed of said codon pairs.

23. A method for expressing a gene in an organism, said gene having altered translational kinetics, comprising the steps of:

introducing a gene prepared according to the method of claim 19 into said organism; and

expressing said gene in said organism.

24. A method for expressing a gene in an organism, said gene having altered translational kinetics,

introducing a gene prepared according to the method of claim 20 into said organism;

and

expressing said gene in said organism.

25. A heterologous DNA sequence coding for a predetermined polypeptide for expression in a first organism, comprising:

a plurality of codon pairs coding for amino acid pairs of acid polypeptide, wherein at least three of said pairs have been selected from among possible codon pairs to correspond to nonrandom codon pair usage patterns of genes expressed at a level within a predetermined range in said first organism.

26. The DNA sequence of claim 25, wherein said predetermined range of expression is levels greater than about 10,000 copies per cell.

27. The DNA sequence of claim 25, wherein said predetermined range of expression is between about 1000 and 10,000 copies per cell.

28. The DNA sequence of claim 25, wherein said predetermined range of expression is below about 1000 copies per cell.

29. A DNA sequence altered for expression in a host organism other than the one to which it is native, wherein at least three codon pairs of the native sequence have been changed to codon pairs preferred by said host organism in genes expressed at a predetermined level.

30. The sequence of claim 29, wherein said alterations conform the pattern of translational kinetics of said altered sequence in said host organism to that of said native sequence in said native organism.

31. An organism expressing an exogenous gene native to a different species, wherein said exogenous gene has been tailored for expression in said organism by rational replacement of existing codon pairs with codon pairs producing desired translational kinetics in said organism.

32. The organism of claim 31, wherein said replacement condon pairs are those that, in comparison to random codon pair usage, are under-represented in said organism.

33. The organism of claim 31, wherein said replacement codon pairs are those that, in comparison to random condon pair usage, are over-represented in said organism.

34. A method for determining the type of organism from which a sample of nucleic acid originated, comprising:

determining the nucleotide sequence of said sample; and

comparing the codon pairings in said sequence with codon pairing preferences for several possible organisms.

35. A method for determining the evolutionary origin of a nucleic acid sample, comprising the steps of:

determining the nucleotide sequence of said sample; and

comparing the codon pairings in said sequence with codon pairing preferences of genes to which the sample may be related.

36. A method for altering the translational kinetics of only a portion of a gene, comprising the steps of:

obtaining, for an organism in which said gene is to be expressed, data regarding the relationship between codon pairing and translational kinetics; and

altering at least one codon pairing in said portion in accordance with said data.

37. The method of claim 36, wherein said alteration introduces a translational pause in said portion.

38. The method of claim 36, wherein said alteration increases the speed with which said portion is translated.

39. A method for controlling protein folding during expression of an exogenous gene in an engineered organism, comprising the steps of:

obtaining data relating to codon pair preferences and relative representation of codon pairs for both a first organism to which said gene is native and a second organism in which said gene is to be expressed;

identifying at least one codon pair in said gene that is over- or under-represented to a different extent in said first organism than in said second organism; and

altering said identified codon pair to a pair coding for the same amino acid that is represented to generally the same degree in said second organism as the identified pair is in said first organism.

40. The method of claim 39, wherein said identified codon pair is over-represented to a greater degree in said first organism than in said second organism.

41. The method of claim 39, wherein said identified codon pair is under-represented to a greater degree in said first organism than in said second organism.

42. The method of claim 39, further comprising the step of expressing said gene in an organism.

43. A method for introducing a translational pause site into a gene to be expressed in a particular organism, comprising the steps of:

obtaining, for said organism, information regarding relative representation of codon pairs; and

introducing into said desired pause site an over-represented codon pair.

44. The method of claim 43, wherein said over-represented codon pair is also at least one standard deviation less abundant than the mean value for codon pairs in said organism.