Variable aberrant cDNAs in single diphtheria toxin-resistant human fibroblasts

Area of Science:

• Molecular Biology
• Genetics
• Cell Biology

Background:

• Diphtheria toxin (DT) exerts its toxic effect by inhibiting protein synthesis.
• Elongation factor 2 (EF-2) is the specific target of DT's ADP-ribosylating activity.
• Understanding mechanisms of toxin resistance is crucial for cellular defense studies.

Purpose of the Study:

• To investigate the molecular basis of diphtheria toxin resistance in transformed human fibroblasts.
• To identify genetic alterations associated with DT resistance.
• To explore the role of aberrant transcripts in conferring toxin resistance.

Main Methods:

• Treatment of transformed human fibroblasts with diphtheria toxin (DT).
• Isolation and characterization of DT-resistant single cells.
• Analysis of transcripts from resistant cells, including cDNA synthesis and deletion mapping.
• Identification of repeat sequences at deletion breakpoints.

Main Results:

• Forty DT-resistant, non-propagating single cells were isolated.
• Thirteen resistant cells exhibited aberrant transcripts of the EF-2 gene.
• cDNA derived from these transcripts contained deletions ranging from 164-447 base pairs.
• Deletions were associated with short repeat sequences (2-8 bp) at their breakpoints, and some deletions involved presumed exon junctions.

Conclusions:

• Aberrant EF-2 transcripts are associated with DT resistance in human fibroblasts.
• The identified deletions in EF-2 cDNA suggest a role for errors during transcription.
• These transcription errors may lead to the production of altered EF-2, conferring resistance to diphtheria toxin.