Screening and use of agents which block or activate intein splicing utilizing natural or homologous exteins

A method of screening for agents which inhibit protein splicing, said method comprising the steps of:

• (a) creating a positive selection system comprising a host cell containing a gene encoding a dominantly cytotoxic protein containing an intein;
• and
• (b) culturing the host cell of step (a) in the presence of test agents, wherein the inhibition of splicing of said cytotoxic protein results in viability of said host cell.

A method for screening of agents which activate protein splicing, said method comprising the steps of:

• (a) creating a positive selection system comprising a host cell containing a gene encoding a dominantly cytotoxic protein, said gene containing an inactive intein;
• and
• (b) culturing said host cell of step (a) in the presence of individual test agents, wherein the activation of splicing of said cytotoxic protein results in host cell death.

A positive genetic selection system for the screening of agents which inhibit protein splicing, said selection system comprising:

• (1) a host cell which contains a first gene encoding a controllable form of a target enzyme which is required for cell viability, and (2) a second gene encoding an expressed form of said target enzyme, said second gene containing an intein, wherein the inhibition of splicing of said target enzyme by a given agent results in the reduced viability or death of said host cell under conditions which do not permit the expression of said controllable first gene of said target enzyme.

A positive genetic selection system for the screening of agents which activate protein splicing, said selection system comprising:

• (1) a host cell which contains a first gene encoding a controllable form of a target enzyme required for cell viability, and (2) a second gene encoding an expressed form of said target enzyme, said second gene containing an inactive intein, wherein the activation of splicing of said target enzyme by a given agent results in the survival of said host cell under conditions which do not permit the expression of said controllable first gene of said target enzyme.

A reporter system for the screening of agents which inhibit protein splicing, said reporter system comprising a host cell which contains a reporter gene encoding a non-essential protein, said reporter gene containing an intein, wherein said intein is selected from the group consisting of a foreign intein inserted into the homologous or non-homologous extein of said reporter gene in the absence of a silent mutation of said extein and a natural intein in a native reporter extein, and wherein the inhibition of splicing of said non-essential protein by a given agent results in a specific detectable phenotype of said host cell.

A reporter system for the screening of agents which activate protein splicing, said reporter system comprising a host cell which contains a reporter gene encoding a non-essential protein, said reporter gene containing an inactive intein, wherein said inactive intein is selected from the group consisting of a foreign inactive intein inserted into the homologous or non-homologous extein of said reporter gene in the absence of a silent mutation of said extein and a natural inactive intein in a native reporter extein, and wherein the activation of splicing of said non-essential protein by a given agent results in a specific selectable phenotype of said host cell.

A method of controlling gene expression in vivo, said method comprising the steps of:

• (a) replacing a homologous extein gene in a host cell with a gene containing an intein;
• and
• (b) modulating the splicing of the intein-containing gene of step (a) with agents which inhibit or activate said splicing.

A method of screening for temperature-sensitive inteins, said method comprising the steps of

• (a) creating a positive selection system comprising a host cell containing a gene encoding a dominantly cytotoxic protein, said gene containing an intein, and wherein said intein is mutagenized;
• and
• (b) culturing the host cell of step (a) at a range of temperatures, wherein at a predetermined temperature, the protein fails to splice and results in viability of said host cell.

A method of screening for temperature-sensitive inteins, said method comprising the steps of

• (a) creating a positive selection system comprising a host cell containing a gene encoding a dominantly cytotoxic protein, said gene containing an inactive intein, and wherein said intein is mutagenized;
• and
• (b) culturing the host cell of step (a) at a range of temperatures, wherein at a predetermined temperature, the intein splices and results in the death of the host cell.

A positive selection system for the screening of temperatures which inhibit protein splicing in temperature-sensitive inteins, said selection system comprising (1) a host cell which contains a first gene encoding a controllable form of a target enzyme which is required for cell viability, and (2) a second gene encoding an expressed form of said target enzyme, said second gene containing an intein, wherein said intein is mutagenized, and wherein the inhibition of splicing of said target enzyme by a given non-permissive temperature results in the reduced viability or death of said host cell under conditions which do not permit the expression of said controllable first gene of said target enzyme.

A positive selection system for the screening of temperatures which activate protein splicing in temperature-sensitive inteins, said selection system comprising (1) a host cell which contains a first gene encoding a controllable form of a target enzyme which is required for cell viability, and (2) a second gene encoding an expressed form of said target enzyme, said second gene containing an intein, wherein said intein is mutagenized, and wherein the activation of splicing of said target enzyme by a given permissive temperature results in the viability of said host cell under conditions which do not permit the expression of said controllable first gene of said target enzyme.

A reporter system for the screening of temperatures which inhibit protein splicing in temperature-sensitive inteins, said reporter system comprising a host cell which contains a reporter gene encoding a non-essential protein, said reporter gene containing an intein, wherein said intein is selected from the group consisting of a foreign intein inserted into the homologous or non-homologous extein of said reporter gene in the absence of a silent mutation of said extein and a natural intein in a native reporter extein, and wherein said intein is mutagenized, and wherein the inhibition of splicing of said non-essential protein by a non-permissive temperature results in a specific detectable phenotype.

A reporter system for the screening of temperatures which activate protein splicing in temperature-sensitive inteins, said reporter system comprising a host cell which contains a reporter gene encoding a non-essential protein, said reporter gene containing an intein, wherein said intein is selected from the group consisting of a foreign intein inserted into the homologous or non-homologous extein of said reporter gene in the absence of a silent mutation of said extein and a natural intein in a native reporter extein, and wherein said intein is mutagenized, and wherein the activation of splicing of said non-essential protein by a permissive temperature results in a specific detectable phenotype.

A positive genetic selection system for screening of agents which inhibit protein splicing, said selection system comprising:

• i) a host which contains a first gene encoding an inactivated form of a target enzyme which is required for cell viability under predetermined conditions;
• and
• ii) a second gene encoding an expressed form of said target enzyme, said second gene containing an intein, wherein the inhibition of splicing of said target enzyme by a given agent results in the reduced viability of said host cell under said predetermined conditions wherein expression of said target enzyme is required for viability or growth.

A positive selection system for the screening of temperatures which inhibit protein splicing in temperature sensitive inteins, said selection system comprising:

• i) a host cell which contains a first gene encoding an inactivated form of the target enzyme which is required for cell viability under predetermined conditions;
• and
• ii) a second gene encoding an expressed form of said target enzyme, said second gene containing an intein, wherein said intein is mutagenized and wherein the inhibition of splicing of said target enzyme at one or more predetermined temperatures results in the reduced viability of said host cell under said predetermined conditions wherein expression of said target enzyme is required for viability or growth.

A method of identifying an agent for antimicrobial activity against a microbial pathogen that naturally has an intein in an essential gene comprising screening for agents that block splicing of that intein in its native context or a homologous extein context.

A method of identifying an agent for antimicrobial activity against a microbial pathogen that naturally has an intein in an essential gene comprising screening for agents that block splicing of that intein in a heterologous extein context that includes one or more native extein residues flanking one or both ends of the intein.

A method of identifying agents with antimicrobial activity against Mycobacterium tuberculosis comprising screening for agents that inhibit splicing of the Mycobacterium tuberculosis DnaB intein.

A method of identifying agents with antimicrobial activity against Mycobacterium leprae comprising screening for agents that inhibit splicing of the Mycobacterium xenopi or Mycobacterium leprae GyrA inteins.

A method of identifying lead compounds with antimicrobial activity comprising identifying agents that inhibit splicing of an intein which is naturally present in that organism.