Cloning of cDNA coding for connective tissue activating peptide III from a human platelet-derived lambda gt11...

Area of Science:

• Molecular Biology
• Hematology
• Protein Chemistry

Background:

• Platelet connective tissue-activating peptide-III (CTAP-III) is an alpha-granule protein found in human platelets.
• Platelet factor 4 (PF4) is another well-characterized platelet alpha-granule protein.
• Understanding the molecular basis of platelet protein synthesis and localization is crucial for hematology research.

Purpose of the Study:

• To clone the complementary DNA (cDNA) encoding human platelet connective tissue-activating peptide-III (CTAP-III).
• To analyze the deduced amino acid sequence of CTAP-III, including its leader sequence.
• To investigate the expression of CTAP-III mRNA in platelets and megakaryocytes.

Main Methods:

• Construction and screening of a lambda gt11 expression library using human platelet messenger RNA (mRNA).
• DNA sequencing of the cloned CTAP-III cDNA.
• Northern blot hybridization to analyze mRNA species in platelets and megakaryocytes.

Main Results:

• Successful cloning of a cDNA encoding a 128-amino acid protein, identified as CTAP-III.
• The 5'-translated region of the cDNA codes for a 34-amino acid leader sequence.
• The leader sequence and mature CTAP-III show significant homology to Platelet Factor 4 (PF4).
• Northern blot analysis revealed a single mRNA species of approximately 0.8 kb in platelets and megakaryocytes.

Conclusions:

• The cloned cDNA is likely full-length and represents the complete coding sequence for CTAP-III.
• The identified leader sequence may play a critical role in targeting CTAP-III to platelet alpha-granules.
• The successful cloning of CTAP-III further validates the specificity of the platelet cDNA library used in this study.