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Cloning of a rabbit erythroid-cell-specific lipoxygenase mRNA
B J Thiele1, J Fleming, K Kasturi
1Beatson Institute for Cancer Research, Glasgow, U.K.
Abstract:
We report the isolation of cDNA recombinants representing part of the rabbit reticulocyte (immature red blood cell, RBC) lipoxygenase (LOX) mRNA. One cDNA predicts an amino acid (aa) sequence matching exactly the unique N-terminal 30-aa sequence of the purified enzyme. Further, the reticulocyte mRNA, hybrid-selected by this recombinant, can be translated in vitro to give a polypeptide that comigrates with the purified reticulocyte LOX and is recognized by affinity-purified anti-RBC LOX polyclonal antibodies. Southern blotting experiments hybridising the RBC LOX cDNAs available to total rabbit genomic DNA digested with various restriction enzymes gives a fairly simple hybridisation pattern under moderate stringency conditions: moreover, the same pattern is obtained with a cloned fragment of genomic DNA containing the RBC LOX gene. This indicates that the RBC LOX gene is unique in the genome and seems not to be very closely related to the genes encoding the other tissue LOXs. We also show by Northern transfer/hybridisation experiments that the RBC LOX mRNA is expressed only in the red cell lineage but not in white blood cells (bone marrow or spleen) or in other non-erythroid cells tested (e.g., brain and lung).
Insights
Researchers isolated rabbit reticulocyte lipoxygenase (LOX) cDNA, confirming its unique gene and expression solely in red blood cells. This finding advances understanding of red blood cell specific gene expression.
Area of Science:
- Molecular Biology
- Hematology
- Gene Expression
Background:
- Lipoxygenases (LOX) are enzymes involved in various cellular processes.
- Understanding the specific LOX expressed in reticulocytes (immature red blood cells) is crucial for hematology research.
- Previous knowledge on the genetic basis and expression patterns of reticulocyte LOX was limited.
Purpose of the Study:
- To isolate and characterize cDNA recombinants for rabbit reticulocyte lipoxygenase (LOX).
- To investigate the genetic uniqueness and expression profile of the reticulocyte LOX gene.
- To confirm the identity and function of the isolated cDNA through in vitro translation and antibody recognition.
Main Methods:
- Isolation of cDNA recombinants from rabbit reticulocyte mRNA.
- In vitro translation of hybrid-selected mRNA.
- Southern and Northern blotting for genomic DNA and mRNA hybridization analysis.
- Amino acid sequence comparison and antibody-based protein detection.
Main Results:
- A cDNA clone was isolated, predicting an N-terminal amino acid sequence matching the purified reticulocyte LOX.
- In vitro translation of hybrid-selected mRNA produced a polypeptide identical to purified reticulocyte LOX.
- Southern blotting indicated a unique reticulocyte LOX gene in the rabbit genome, distinct from other tissue LOX genes.
- Northern blotting revealed that reticulocyte LOX mRNA is exclusively expressed in the red blood cell lineage.
Conclusions:
- The study successfully isolated and characterized cDNA for rabbit reticulocyte lipoxygenase.
- The reticulocyte LOX gene is unique and specifically expressed in erythroid cells, not in other tissues or white blood cells.
- These findings provide a molecular basis for understanding red blood cell-specific gene expression and the role of LOX in erythropoiesis.