Related Experiment Videos
Cloning and characterization of CneMDR1: a Cryptococcus neoformans gene encoding a protein related to multidrug
S J Thornewell1, R B Peery, P L Skatrud
1Infectious Diseases Research, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN 46285, USA.
Abstract:
CneMDR1, a gene encoding a protein related to several eukaryotic multidrug resistance (MDR) proteins, was identified, cloned, and characterized from a clinical isolate of Cryptococcus neoformans (Cn) (strain M1-106). Polymerase chain reaction (PCR) amplification of a DNA region encompassing conserved motifs of other MDR-like proteins was initially used to identify and clone CneMDR1. Analysis of the corresponding cDNA revealed an open reading frame punctuated by 16 introns. CneMDR1 encoded a protein (CNEMDR1) containing 1408 amino acids (aa) with a predicted mass of approximately 152kDa. Protein structure predictions suggested the presence of two putative 6-transmembrane (TM) domains as well as two ATP-binding domains, structural characteristics typical of ATP-binding cassette (ABC) proteins. Members of this superfamily, which include MDR proteins, are frequently involved in active transport of a variety of substrates across the cell membrane. Pulsed-field gel electrophoresis revealed the presence of 12 chromosomal bands in this clinical isolate of Cn. CneMDR1 was detected by hybridization on chromosome IV. High-stringency hybridization detected only one MDR-like gene. However, a second MDR-like gene (CneMDR2) was discovered during reverse transcriptase-PCR (RT-PCR) amplification using cDNA.
Insights
Researchers identified and cloned the CneMDR1 gene from Cryptococcus neoformans, revealing a protein with structural similarities to eukaryotic multidrug resistance (MDR) proteins. This discovery sheds light on potential drug resistance mechanisms in this fungal pathogen.
Area of Science:
- Mycology
- Molecular Biology
- Biochemistry
Background:
- Cryptococcus neoformans is a fungal pathogen responsible for cryptococcosis.
- Multidrug resistance (MDR) proteins, often ATP-binding cassette (ABC) transporters, play a crucial role in cellular defense and drug resistance.
- Understanding the genetic basis of drug resistance in C. neoformans is vital for developing effective treatments.
Purpose of the Study:
- To identify, clone, and characterize a novel multidrug resistance gene from a clinical isolate of Cryptococcus neoformans.
- To elucidate the structural and functional characteristics of the identified gene and its encoded protein.
- To investigate the presence and location of MDR-like genes within the C. neoformans genome.
Main Methods:
- Polymerase chain reaction (PCR) amplification using conserved motifs of MDR-like proteins.
- cDNA analysis to determine the open reading frame, intron-exon structure, and protein characteristics.
- Protein structure prediction to identify transmembrane and ATP-binding domains.
- Pulsed-field gel electrophoresis (PFGE) for chromosomal analysis.
- Southern hybridization for gene localization.
- Reverse transcriptase-PCR (RT-PCR) for gene discovery.
Main Results:
- The CneMDR1 gene was successfully identified and cloned from Cryptococcus neoformans.
- CneMDR1 encodes a protein (CNEMDR1) of 1408 amino acids with predicted 6-transmembrane and 2 ATP-binding domains, characteristic of ABC transporters.
- Hybridization studies localized CneMDR1 to chromosome IV.
- A second MDR-like gene, CneMDR2, was identified using RT-PCR, suggesting the presence of multiple MDR-like genes.
Conclusions:
- CneMDR1 is a novel MDR-like gene in Cryptococcus neoformans, belonging to the ABC transporter superfamily.
- The structural features of CNEMDR1 suggest a role in active transport, potentially contributing to drug resistance.
- The identification of CneMDR1 and CneMDR2 provides insights into the genetic mechanisms underlying multidrug resistance in this important fungal pathogen.