Temperature-sensitive mutations made easy: generating conditional mutations by using temperature-sensitive inteins that function within different temperature ranges.

نویسندگان

  • Guihong Tan
  • Ming Chen
  • Christopher Foote
  • Change Tan
چکیده

Reversible and easy to use, temperature-sensitive (TS) mutations are powerful tools for studying gene function. However, TS alleles are rare and difficult to generate and identify, and this has limited their use in most multicellular organisms. We have generated and characterized 41 intein switches, temperature-sensitive Sce VMA mutations that splice only at the permissive temperatures to generate intact host proteins. At nonpermissive temperatures, they fail to splice, resulting in a loss of function of the proteins in which they reside. By inserting an intein switch into a protein of interest, one can turn on and off the activities of the engineered protein with a simple temperature shift. The 41 TS inteins function in five different temperature ranges, with permissive temperatures ranging from 18 degrees to 30 degrees . This collection makes it possible to choose a TS-intein switch according to the optimal growth temperature of an organism or to suit a special experimental design.

برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید

ثبت نام

اگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید

منابع مشابه

Construction of the Recombinant Plasmid Expressing AID under the Control of Temperature-sensitive Promoter of Bacteriophage Lambda

Background and purpose: Activation-induced cytidine deaminase (AID) is a B-cell specific enzyme responsible for somatic hypermutation (SHM) and class switch recombination (CSR) of antibody genes within the B-cell follicle of peripheral lymphoid organs. Ectopic overexpression of the enzyme leads to mutations in non-B cells and Escherichia coli (E.coli) genes. However, induction of mutations in E...

متن کامل

Development of a positive genetic selection system for inhibition of protein splicing using mycobacterial inteins in Escherichia coli DNA gyrase subunit A.

An intein-based positive genetic selection system was developed to study protein splicing and to provide a selection system with the potential for finding splicing inhibitors. Inteins can be novel antimicrobial targets when present in essential proteins since blocking splicing would kill the organism. For example, pathogenic mycobacteria encode inteins that interrupt DNA gyrase. The gyrase sele...

متن کامل

Temperature-sensitive mutations of the notch locus in Drosophila melanogaster.

Temperature-conditional mutations of the Notch locus were characterized in an attempt to understand the organization of a "complex locus" and the control of its function in development. Among 21 newly induced Notch alleles, about one-half are temperature-conditional for some effects, and three are temperature-sensitive for viability. One temperature-sensitive lethal, l(1)Nts1, is functionally n...

متن کامل

A Survey of New Temperature-Sensitive, Embryonic-Lethal Mutations in C. elegans: 24 Alleles of Thirteen Genes

To study essential maternal gene requirements in the early C. elegans embryo, we have screened for temperature-sensitive, embryonic lethal mutations in an effort to bypass essential zygotic requirements for such genes during larval and adult germline development. With conditional alleles, multiple essential requirements can be examined by shifting at different times from the permissive temperat...

متن کامل

ذخیره در منابع من


  با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید

برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید

ثبت نام

اگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید

عنوان ژورنال:
  • Genetics

دوره 183 1  شماره 

صفحات  -

تاریخ انتشار 2009