Related Experiment Videos
Antigen-specific T cell suppressor factor (TsF): isolation of a cDNA clone encoding for a functional polypeptide
Abstract:
A cDNA library of phosphorylcholine (PC)-specific suppressor T hybridoma, 29-C-6, was constructed. By differential colony hybridization, 68 colonies were selected which hybridized with mRNA probes of two PC-specific suppressor T hybridomas, 29-C-6 and 09-M-24-8, but not with those of BW5147. A hybridization translation assay revealed that only one combination of translation products of cDNA clones, p6-1 and p6-5, showed strong PC-T cell suppressor factor (TsF) activity. Sequence analysis showed that p6-5 contained a specific cDNA sequence of about 800 base pairs (bp) while p6-1 had a 190 bp poly(A) sequence insert. When total poly(A) + RNA of 29-C-6 was hybridized with p6-1 filter alone the recovered mRNA was capable of producing PC-TsF. However, when the total poly-(A) + RNA was hybridized with a p6-1 filter combined with a p6-5 filter, the mRNA eluted from the p6-1 filter was not able to produce PC-TsF, suggesting that the depletion of p6-5 specific mRNA from p6-1-binding poly(A) + RNA led to a complete abolishment of the capability to produce PC-TsF. Furthermore, p6-5 hybridizing mRNA could successfully restore the p6-1-binding poly(A) + RNA depleted of p6-5-specific mRNA, and translation products of both RNA mixtures showed strong PC-TsF activity. These results suggest that PC-TsF is not a single polypeptide chain, but is composed of at least two distinct polypeptide chains, and also that p6-5 contains a cDNA sequence encoding for one of the polypeptide chains composing the PC-TsF molecule.
Insights
This study identified that phosphorylcholine (PC)-specific T cell suppressor factor (TsF) is composed of at least two distinct polypeptide chains. The research isolated cDNA clones encoding these components, revealing the molecular basis of TsF activity.
Area of Science:
- Immunology
- Molecular Biology
- Biochemistry
Background:
- Suppressor T cells play a crucial role in immune regulation.
- Phosphorylcholine (PC)-specific suppressor T cell factor (TsF) activity needs further molecular characterization.
Purpose of the Study:
- To identify and characterize the molecular components of PC-specific TsF.
- To elucidate the subunit composition of the PC-TsF molecule.
Main Methods:
- Construction of a cDNA library from PC-specific suppressor T hybridoma 29-C-6.
- Differential colony hybridization to screen for relevant cDNA clones.
- Hybridization-translation assays to assess TsF activity of translation products.
Main Results:
- Identified cDNA clones p6-1 and p6-5 whose combined translation products exhibited PC-TsF activity.
- Demonstrated that p6-5 specific mRNA depletion abolished TsF activity, which could be restored by adding back p6-5 mRNA.
- Sequence analysis indicated p6-5 contains an 800 bp cDNA sequence, while p6-1 contains a poly(A) sequence insert.
Conclusions:
- PC-TsF is composed of at least two distinct polypeptide chains.
- cDNA clone p6-5 encodes one of the polypeptide chains essential for PC-TsF activity.