Hybrid nucleic acid sequences having tandemly arranged genes

Claim 1

A hybrid nucleic acid sequence for use in producing one or more selected polypeptides in a host cell comprising: a promoter sequence recognized by the host cell at one end; a transcription terminator sequence recognized by the host cell at the other end; and between the promoter sequence and the transcription terminator sequence: a first region which includes a ribosome-binding site, a translation initiation site, a coding region for one of said one or more selected polypeptides, and a translation termination site, but which does not include the promoter sequence or the transcription terminator sequence; and one or more second regions, each of which includes a ribosome-binding site, a translation initiation site, a coding region for the same or a different one of said one or more selected polypeptides, and a translation termination site, and which does not include the promoter sequence or the transcription terminator sequence, the reading directions of the promoter sequence, the transcription terminator sequence, the first region and each of the second regions having the same orientation.

Claim 6

A method for synthesizing one or more selected polypeptides in a microorganism comprising the steps of: (a) transforming the microorganism with a hybrid nucleic acid sequence comprising: a promoter sequence recognized by the microorganism at one end; a transcription terminator sequence recognized by the host cell at the other end; and between the promoter sequence and the transcription terminator sequence: a first region which includes a ribosome-binding site, a translation initiation site, a coding region for one of said one or more selected polypeptides, and a translation termination site, but which does not include the promoter sequence or the transcription terminator sequence; and one or more second regions, each of which includes a ribosome-binding site, a translation initiation site, a coding region for the same or a different one of said one or more selected polypeptides, and a translation termination site, and which does not include the promoter sequence or the transcription terminator sequence, the reading directions of the promoter sequence, the transcription terminator sequence, the first region and each of the second regions having the same orientation; (b) culturing the transformed microorganism; and (c) collecting the one or more selected polypeptides.

Claim 7

A method for synthesizing one or more selected polypeptides in a microorganism comprising the steps of: (a) transforming the microorganism with a hybrid nucleic acid sequence comprising: a promoter sequence recognized by the microorganism at one end; a transcription terminator sequence recognized by the host cell at the other end; and between the promoter sequence and the transcription terminator sequence: a first region which includes a ribosome-binding site, a translation initiation site, a coding region for one of said one or more selected polypeptides, and a translation termination site, but which does not include the promoter sequence or the transcription terminator sequence; and one or more second regions, each of which includes a ribosome-binding site, a translation initiation site, a coding region for the same or a different one of said one or more selected polypeptides, and a translation termination site, and which does not include the promoter sequence or the transcription terminator sequence, the reading directions of the promoter sequence, the transcription terminator sequence, the first region and each of the second regions having the same orientation; (b) culturing the transformed microorganism; and (c) collecting the one or more selected polypeptides.

Claim 10

A method for producing a hybrid nucleic acid sequence wherein the coding regions for one or more selected polypeptides are tandemly arranged and placed under the control of a common promoter sequence and a common transcription terminator sequence, comprising the steps of: (a) preparing or selecting first nucleic acid sequences which do not include the promoter sequence or the transcription terminator sequence, which have ligatable ends, and which include: (1) a region which codes for one of the one or more selected polypeptides; (2) a translation initiation site; (3) a translational termination site; and (4) a ribosome-binding site; (b) ligating the first nucleic acid sequences to form second nucleic acid sequences which have ligatable ends and which include tandemly arranged coding regions for the one or more selected polypeptides; (c) preparing or selecting third nucleic acid sequences which have ligatable ends and which include the promoter sequence and the transcription terminator sequence; (d) ligating the second nuclei acid sequences with the third nucleic acid sequences to form fourth nucleic acid sequences; and (e) selecting those fourth nucleic acid sequences in which each of the regions coding for one of said one or more selected polypeptides, each of the translation initiation sites, each of the translation termination sites, each of the ribosome-binding sites, the promoter sequence, and the transcription terminator sequence have the same reading direction.

Claim 11

A method for producing a hybrid nucleic acid sequence wherein the coding regions for one or more selected polypeptides are tandemly arranged and placed under the control of a common promoter sequence and a common transcription terminator sequence, comprising the steps of: (a) preparing or selecting first nucleic acid sequences which do not include the promoter sequence or the transcription terminator sequence, which have ligatable ends, and which include: (1) a region which codes for one of the one or more selected polypeptides; (2) a translation initiation site; (3) a translational termination site; and (4) a ribosome-binding site; (b) ligating the first nucleic acid sequences to form second nucleic acid sequences which have ligatable ends and which include tandemly arranged coding regions for the one or more selected polypeptides; (c) preparing or selecting third nucleic acid sequences which have ligatable ends and which include the promoter sequence and the transcription terminator sequence; (d) ligating the second nuclei acid sequences with the third nucleic acid sequences to form fourth nucleic acid sequences; and (e) selecting those fourth nucleic acid sequences in which each of the regions coding for one of said one or more selected polypeptides, each of the translation initiation sites, each of the translation termination sites, each of the ribosome-binding sites, the promoter sequence, and the transcription terminator sequence have the same reading direction.

Claim 15

A method for combining nucleic acid sequences so that their reading directions have the same orientation, comprising the steps of: (a) preparing or selecting sequences which at one end have a ligatable end of a first type and at the other end have a ligatable end of a second type, the reading direction of a sequence being from the ligatable end of the first type towards the ligatable end of the second type, the union of two ligatable ends of the first type being susceptible to cleavage by a first restriction enzyme, the union of two ligatable ends of the second type being susceptible to cleavage by a second restriction enzyme, the union of .a ligatable end of the first type with a ligatable end of the second type not being substantially susceptible to cleavage by either the first or the second restriction enzyme; (b) ligating the sequences; and (c) treating the ligated sequences with the first and second restriction enzymes.

Claim 16

A method for combining nucleic acid sequences so that their reading directions have the same orientation, comprising the steps of: (a) preparing or selecting sequences which at one end have a ligatable end of a first type and at the other end have a ligatable end of a second type, the reading direction of a sequence being from the ligatable end of the first type towards the ligatable end of the second type, the union of two ligatable ends of the first type being susceptible to cleavage by a first restriction enzyme, the union of two ligatable ends of the second type being susceptible to cleavage by a second restriction enzyme, the union of .a ligatable end of the first type with a ligatable end of the second type not being substantially susceptible to cleavage by either the first or the second restriction enzyme; (b) ligating the sequences; and (c) treating the ligated sequences with the first and second restriction enzymes.

Claim 17

A method for combining nucleic acid sequences of a first type with nucleic acid sequences of a second type to form hybrid sequences in which the reading directions of the the first and second sequences have the same orientation, and in which the number of first and second sequences are approximately equal, comprising the steps of: (a) preparing or selecting sequences of the first type which at one end have a ligatable end of a first type and at the other end have a ligatable end of a second type, the reading direction of a sequence being from the ligatable end of the first type towards the ligatable end of the second type, the ligatable end of the first type not being substantially ligatable with the ligatable end of the second type, the union of two ligatable ends of the first type being susceptible to cleavage by a first restriction enzyme, the union of two ligatable ends of the second type being susceptible to cleavage by a second restriction enzyme; (b) preparing or selecting sequences of the second type which at one end have a ligatable end of a third type and at the other end have a ligatable end of a fourth type, the reading direction of a sequence being from the ligatable end of the third type towards the ligatable end of the fourth type, the ligatable end of the third type not being substantially ligatable with the ligatable end of the fourth type, the ligatable end of the first type not being substantially ligatable with the ligatable end of the third type, the ligatable end of the second type not being substantially ligatable with the ligatable end of the fourth type, the union of two ligatable ends of the third type being susceptible to cleavage by a third restriction enzyme, the union of two ligatable ends of the fourth type being susceptible to cleavage by a fourth restriction enzyme, the union of a ligatable end of the first type with a ligatable end of the fourth type and the union of a ligatable end of the second type with a ligatable end of the third type not being substantially susceptible to cleavage by any of the first, second, third or fourth restriction enzymes; (c) ligating the sequences of the first and second types with each other; and (d) treating the ligated sequences with the first, second, third and fourth restriction enzymes.

Claim 18

A method for combining nucleic acid sequences of a first type with nucleic acid sequences of a second type to form hybrid sequences in which the reading directions of the the first and second sequences have the same orientation, and in which the number of first and second sequences are approximately equal, comprising the steps of: (a) preparing or selecting sequences of the first type which at one end have a ligatable end of a first type and at the other end have a ligatable end of a second type, the reading direction of a sequence being from the ligatable end of the first type towards the ligatable end of the second type, the ligatable end of the first type not being substantially ligatable with the ligatable end of the second type, the union of two ligatable ends of the first type being susceptible to cleavage by a first restriction enzyme, the union of two ligatable ends of the second type being susceptible to cleavage by a second restriction enzyme; (b) preparing or selecting sequences of the second type which at one end have a ligatable end of a third type and at the other end have a ligatable end of a fourth type, the reading direction of a sequence being from the ligatable end of the third type towards the ligatable end of the fourth type, the ligatable end of the third type not being substantially ligatable with the ligatable end of the fourth type, the ligatable end of the first type not being substantially ligatable with the ligatable end of the third type, the ligatable end of the second type not being substantially ligatable with the ligatable end of the fourth type, the union of two ligatable ends of the third type being susceptible to cleavage by a third restriction enzyme, the union of two ligatable ends of the fourth type being susceptible to cleavage by a fourth restriction enzyme, the union of a ligatable end of the first type with a ligatable end of the fourth type and the union of a ligatable end of the second type with a ligatable end of the third type not being substantially susceptible to cleavage by any of the first, second, third or fourth restriction enzymes; (c) ligating the sequences of the first and second types with each other; and (d) treating the ligated sequences with the first, second, third and fourth restriction enzymes.