Beta-amyloid peptdie-binding proteins and polynucleotides encoding the same
A technology of polynucleotide and amyloid peptide, applied in the field of protein
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0099] Example 1: Cloning and isolation of BAP binding protein (BBP1)
[0100] A yeast two-hybrid (Y2H) genetic screen was developed to identify genes related to human BAP 42 (A 42-amino acid proteolytic fragment of APP, thought to be the likely more toxic aggregated form of BAP) interacting proteins. BAP 42 Expressed as a fusion with the yeast Gal4 DNA-binding domain and also as a free peptide ( figure 1 ). This strain was transformed with a human fetal brain cDNA Y2H library. from about 10 6 One of the independent transformants (named clone 14 above) produced consistent reporter gene activity and contained an important open reading frame contiguous to the GAL4 domain. The cDNA insert consists of 984 base pairs, terminated by a polyA sequence. The sequence encodes 201 amino acids (SEQ ID NO: 2; amino acid residues 68-269) with two regions of sufficient length and hydrophobicity to span cell membranes.
[0101] A library-derived plasmid was isolated from clone 14 and us...
Embodiment 2
[0102] Example 2: Isolation and confirmation of the 5' end of BBP1
[0103] The BBP1 cDNA sequence contained in clone 14 described in Example 1 above lacked the 5' end of the protein coding region because no potential start codon methionine was present. Multiple attempts at conventional 5' RACE (rapid amplification of cDNA ends) with standard reverse transcriptase added only 27 nucleotides. These sequences included an ATG, but in the same translation reading frame, without an upstream stop codon to believe this was a start codon. A genomic cloning method was initiated to isolate the 5' end of the BBP1 gene.
[0104] The human genomic lambda library was hybridized with a 400 base pair (bp) random primed probe corresponding to the 5' sequence of clone 14, identifying 10 positive clones. Further characterization of these clones with a 45 base nucleotide probe corresponding to the most upstream BBP1 sequence of clone 14 (and corresponding to the 5' upstream sequence of the 400 b...
Embodiment 3
[0106] Example 3: Characteristic analysis of BBP1
[0107] The BBP1 sequence was compared to Genbank using the base local alignment search tool (BLAST; Altschul et al., 1990). Two Caenorhabditis elegans and one Drosophilamelanogaster genome sequences and a large number of human, mouse and other mammalian expressed sequence markers were identified. However, no complete cDNA sequence was obtained, nor functional data conferred by the gene. The sequences of the BBP1 protein and the obtained translation products of expressed sequence tags were compared, conserved segments were searched, and evaluated with MoST (Protein Motif Search Algorithm) (Tatusov et al., 1994). These analyzes revealed potential evolutionary relationships between families of receptors coupled to G proteins. Specifically, these analyzes revealed that BBP1 contains two potential transmembrane (tm) domains that are comparable to tm domains 3 and 4 of G protein-coupled receptors. The central hydrophilic ring co...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More