Molecular Characterization of a Gene Encoding a Mucin-Like Protein from Caenorhabditis Elegans

• Main Author: Ceroni da Silva, Sergio
• Format: Dissertation
• Language: English
• Published: ProQuest Dissertations & Theses 01-01-1993
• Subjects: Proteins

A mutator strain containing a Tc 1 transposon disrupting the dpy-6 gene activity was used to isolate the gene. This strain was backcrossed to the N2 wild-type strain in order to reduce and stabilize the Tc 1 copy number. After six rounds of backcrosses, a single Tc1 insertion associated with the dumpy phenotype could be identified. This Tc 1 insertion was located in a 9.5 kb EcoRI fragment, which was physically mapped, by hybridization to YACs and cosmids, to a region in the X chromosome of C. elegans were the dpy-6 gene is located. The Tc 1 tagged fragment was cloned and the sequence adjacent to the insertion site was determined. An open reading frame containing 1857 by was found, capable of encoding a protein with 619 amino acids. The putative protein was similar to mucin-like sequences, with the presence of thirty four copies of a six amino acid repeat arranged in tandem near the carboxy-terminus. Some of the repeats were also found dispersed in other parts of the putative protein. The amino acid repeats, like in other mucin-like proteins, were rich in threonine, serine and proline. Another aspect which has been investigated was the identification of a collagenolytic activity in C. elegans. Using substrate gels containing collagen, this activity was found associated exclusively with the cuticle material. Attempts were made to isolate the corresponding gene by Polymerase Chain Reaction using oligonucleotides specific for conserved regions within metalloproteases.