Related Experiment Videos
Cloning and sequencing of a human pancreatic tumor mucin cDNA
1Department of Microbiology and Immunology, Duke University Medical Center, Durham, North Carolina 27710.
Abstract:
A monospecific polyclonal antiserum against deglycosylated human pancreatic tumor mucin was used to select human pancreatic mucin cDNA clones from a lambda gt11 cDNA expression library developed from a human pancreatic tumor cell line. The full-length 4.4-kilobase mucin cDNA sequence included a 72-base pair 5'-untranslated region and a 307-base pair 3'-untranslated region. The predicted amino acid sequence for this cDNA revealed a protein of 122,071 daltons containing 1,255 amino acid residues of which greater than 60% were serine, threonine, proline, alanine, and glycine. Approximately two-thirds of the protein sequence consisted of identical 20-amino acid tandem repeats which were flanked by degenerate tandem repeats and nontandem repeat sequences on both the amino-terminal and carboxyl-terminal ends. The amino acid sequence also contained five putative N-linked glycosylation sites, a putative signal sequence and transmembrane domain, and numerous serine and threonine residues (potential O-linked glycosylation sites) outside and within the tandem repeat position. The cDNA and deduced amino acid sequence of the pancreatic mucin sequence was over 99% homologous with a mucin cDNA sequence derived from breast tumor mucin, even though the native forms of these molecules are quite distinct in size and degree of glycosylation.
Insights
Researchers identified human pancreatic mucin cDNA, revealing a protein rich in serine and threonine. This pancreatic mucin shares high homology with breast tumor mucin, despite differences in their native forms.
Area of Science:
- Molecular Biology
- Biochemistry
- Oncology
Background:
- Mucins are high molecular weight glycoproteins implicated in various cancers.
- Human pancreatic tumor mucin (h-P-TIM) structure and genetic basis require further elucidation.
Purpose of the Study:
- To isolate and characterize the complementary DNA (cDNA) encoding human pancreatic tumor mucin.
- To analyze the deduced amino acid sequence and identify key structural features.
Main Methods:
- Construction of a human pancreatic tumor cell line cDNA expression library in lambda gt11.
- Screening the library using a monospecific polyclonal antiserum against deglycosylated human pancreatic tumor mucin.
- Sequencing of the isolated mucin cDNA and analysis of the deduced protein sequence.
Main Results:
- Isolation of a full-length 4.4-kilobase mucin cDNA.
- The deduced protein (122,071 daltons, 1,255 residues) is rich in serine, threonine, proline, alanine, and glycine (>60%).
- The protein contains extensive tandem repeats (20 amino acids), potential N-linked and O-linked glycosylation sites, a signal sequence, and a transmembrane domain.
- Pancreatic mucin cDNA showed >99% homology to breast tumor mucin cDNA.
Conclusions:
- The characterized cDNA provides a molecular basis for human pancreatic mucin.
- The high homology suggests conserved structural domains or evolutionary origins between pancreatic and breast mucins.
- Further investigation into the distinct native forms despite sequence similarity is warranted.