ABSTRACT
The correct identification of all human genes, and their derived transcripts, has not yet been achieved, and it remains one of the major aims of the worldwide genomics community. Computational programs suggest the existence of 30,000 to 40,000 human genes. However, definitive gene identification can only be achieved by experimental approaches. We used two distinct methodologies, one based on the alignment of mouse orthologous sequences to the human genome, and another based on the construction of a high-quality human testis cDNA library, in an attempt to identify new human transcripts within the human genome sequence. We generated 47 complete human transcript sequences, comprising 27 unannotated and 20 annotated sequences. Eight of these transcripts are variants of previously known genes. These transcripts were characterized according to size, number of exons, and chromosomal localization, and a search for protein domains was undertaken based on their putative open reading frames. In silico expression analysis suggests that some of these transcripts are expressed at low levels and in a restricted set of tissues.
Subject(s)
DNA, Complementary/genetics , Genome, Human , Sequence Analysis, DNA/methods , Testis/chemistry , Transcription, Genetic/genetics , Amino Acid Sequence , Animals , Chromosome Mapping , Gene Library , Humans , Male , Mice , Molecular Sequence DataABSTRACT
Pancreatic amyloid plaques formed by the pancreatic islet amyloid polypeptide (IAPP) are present in more than 95% of type II diabetes mellitus patients, and their abundance correlates with the severity of the disease. IAPP is currently considered the most amyloidogenic peptide known, but the molecular bases of its aggregation are still incompletely understood. Detailed characterization of the mechanisms of amyloid formation requires large quantities of pure material. Thus, availability of recombinant IAPP in sufficient amounts for such studies constitutes an important step toward elucidation of the mechanisms of amyloidogenicity. Here, we report, for the first time, the successful expression, purification and characterization of the amyloidogenicity and cytotoxicity of recombinant human mature IAPP. This approach is likely to be useful for the production of other amyloidogenic peptides or proteins that are difficult to obtain by chemical synthesis.
Subject(s)
Amyloid/chemistry , Glycine/analogs & derivatives , Amino Acid Sequence , Amyloid/metabolism , Base Sequence , Cells, Cultured , Cloning, Molecular , DNA, Complementary/metabolism , Dose-Response Relationship, Drug , Electrophoresis, Polyacrylamide Gel , Escherichia coli/metabolism , Glycine/chemistry , Humans , Islet Amyloid Polypeptide , Microscopy, Electron , Microscopy, Fluorescence , Molecular Sequence Data , Mutation , Peptides/chemistry , Protein Structure, Tertiary , Recombinant Fusion Proteins/chemistry , Recombinant Proteins/chemistry , Reverse Transcriptase Polymerase Chain Reaction , Spectrometry, Fluorescence , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Time FactorsABSTRACT
The correct identification of all human genes, and their derived transcripts, has not yet been achieved, and it remains one of the major aims of the worldwide genomics community. Computational programs suggest the existence of 30,000 to 40,000 human genes. However, definitive gene identification can only be achieved by experimental approaches. We used two distinct methodologies, one based on the alignment of mouse orthologous sequences to the human genome, and another based on the construction of a high-quality human testis cDNA library, in an attempt to identify new human transcripts within the human genome sequence. We generated 47 complete human transcript sequences, comprising 27 unannotated and 20 annotated sequences. Eight of these transcripts are variants of previously known genes. These transcripts were characterized according to size, number of exons, and chromosomal localization, and a search for protein domains was undertaken based on their putative open reading frames. In silico expression analysis suggests that some of these transcripts are expressed at low levels and in a restricted set of tissues.