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Molecular cloning, sequencing, and brain expression of the presenilin 1 gene in Microcebus murinus
A Calenda1, N Mestre-Francés, C Czech
1INSERM U249, CNRS UPR9008, Institut de Biologie, Montpellier, France. calenda@xerxes.crbm.cnrs-mop.fr
Abstract:
The cDNA encoding the Microcebus murinus presenilin 1 protein (PSI) was cloned by RT-PCR from a brain cDNA library using various combinations of oligonucleotide primers designed on the basis of the human nucleotide sequence. Analysis of five clones isolated from two positive combinations revealed that the deduced open reading frame encodes two protein isoforms of 467 and 463 amino acid residues. The shorter isoform lacked the four residues VRSQ in the N-terminal region and like the 467 amino acid isoform presented 22 substitutions with its human homologue. The 12 bp nucleotide deletion evidenced in the cDNA encoding the shorter isoform is consistent with the use of an alternative 5' splice donor site identified at the end of the human exon 3. The immunohistochemistry performed with a specific polyclonal antiserum raised against a synthetic peptide located in the human large hydrophilic loop of PS1 revealed that the protein is widely distributed independently of age or of pathology in the microcebe brain. PS1 is found predominantly in neurons of the different cortical layers and hippocampus but also in subcortical structures. The PS1 labelling appeared as thin granulations scattered throughout the cytoplasm of numerous neurons and sometimes in neurites.
Insights
Researchers cloned the Microcebus murinus presenilin 1 protein (PS1) cDNA, identifying two isoforms with variations from the human homolog. Immunohistochemistry revealed widespread PS1 distribution in the microcebe brain, primarily in neurons.
Area of Science:
- Neuroscience
- Molecular Biology
- Genetics
Background:
- Presenilin 1 (PS1) is a key component of the gamma-secretase complex implicated in Alzheimer's disease.
- Understanding PS1 in non-human primates can provide insights into its function and evolution.
Purpose of the Study:
- To clone and characterize the cDNA encoding Microcebus murinus presenilin 1 (PS1).
- To investigate the distribution of PS1 protein in the microcebe brain.
Main Methods:
- Reverse transcription-polymerase chain reaction (RT-PCR) was used to clone the cDNA.
- Sequence analysis identified two protein isoforms.
- Immunohistochemistry was performed using a specific polyclonal antiserum.
Main Results:
- Two PS1 isoforms of 467 and 463 amino acids were identified, with the shorter isoform lacking four N-terminal residues.
- The microcebe PS1 exhibited 22 substitutions compared to its human homolog.
- PS1 was widely distributed in the microcebe brain, predominantly in neurons of cortical layers, hippocampus, and subcortical structures, irrespective of age or pathology.
Conclusions:
- The study successfully cloned and characterized Microcebus murinus PS1, revealing isoform diversity and evolutionary divergence from human PS1.
- PS1 protein is broadly expressed in the microcebe brain, suggesting a fundamental role in neuronal function.