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Biomedical subjects

L A Bejarano

Publications and source records attributed to L A Bejarano.

6 recordsLinked to original sources
PubMed2000

Protein traps: using intracellular localization for cloning.

A much-sought goal - the rapid cloning of genes whose protein products have specific intracellular localizations - has now been made possible. A visual screen of cells expressing fusions between coding DNA sequences and a reporter, such as green-fluorescent protein (GFP) or beta-galactosidase, identifies cells with the pattern of interest. The DNA sequences encoding these targeted fusions can then be cloned either directly from these cells or by repeated rounds of screening and subdivision of library pools. It is expected that systematic screenings based on these methods will identify additional components for every compartment and define new domains, thus facilitating a more comprehensive understanding of the cellular architecture.

Cell Compartmentation↗
PubMed1999

Motif trap: a rapid method to clone motifs that can target proteins to defined subcellular localisations.

We have developed a rapid procedure termed Motif Trap (MT) to identify protein motifs that are able to target proteins to a distinct subcellular localisation in eukaryotic cells. By expressing random DNA fragments fused to green fluorescent protein (GFP), individual cells with the GFP localisation of interest are readily isolated allowing for the expressed DNA fragments to be cloned by RT-PCR. These can then be used to identify the corresponding full-length cDNAs. Using MT, we have identified patterns of GFP localisation which correspond to every major organelle and compartment. We have shown that MT is useful to identify new sequences that determine subcellular localisation as well as known targeting motifs.

Amino Acid Sequence↗
PubMed1996

Molecular cloning of an intronless gene for the hamster centromere antigen CENP-B.

Centromere protein B (CENP-B) is a DNA-binding protein present at both active and inactive centromeres. It was first localized at the kinetochore region by human autoimmune sera from CREST patients. Using a previously identified human cDNA we have isolated a genomic clone containing the complete hamster CENP-b intronless coding sequence. At the nucleotide level it was found to possess a high degree of homology with the human and mouse CENP-B genes, being 75% and 90% respectively. This codes for 606 amino acid residues, which represent seven more than the human and mouse centromeric proteins. Hamster CENP-B protein analysis revealed at the N-terminal region a 133 amino acid fragment of 100% homology to the DNA binding motif identified previously for the human autoantigen. Expression of hamster CENP-B during the cell cycle was analyzed by using a specific antiCENP-B serum generated against the C-terminal conserved region. These data indicate that CENP-B is highly conserved and it represents a universal component of the centromere structure and function in mammals.

Amino Acid Sequence↗
PubMed1993

Anticentromere antibody specific to human cells directed against the CENP-B autoantigen.

We describe the generation of a new antipeptide antibody that binds to the centromeric region of human mitotic chromosomes. This antibody was raised against a synthetic peptide corresponding to the 481-493 amino acid sequence of the human CENP-B autoantigen. Immunofluorescence analysis revealed that this anti-CENP-B serum showed an identical pattern to the human CREST anticentromere autoantibody in both mitotic cells and interphase nuclei. Immunoblotting showed that this antibody reacts with the recombinant human CENP-B autoantigen, indicating that it is directed to the 80-kDa centromere polypeptide. We have used this serum to determine, by indirect immunofluorescence, whether CENP-B is conserved in different mammalian species. Surprisingly, the human antipeptide antibody does not react with the centromeric proteins of cultured mouse, hamster, or Indian muntjac cells. Because the CENP-B gene has been cloned in human and mouse, our results suggest that the CENP-B epitope used as an immunogen in this study is not ubiquitous in mammalian cells, and that we have most probably established a monospecific antibody to the human centromere.

3T3 Cells↗
PubMed1991

Monoclonal antibody with specificity to a conserved epitope in the C-terminal domain of histone H1 variants.

A monoclonal type M-immunoglobulin (IgM) was generated in mice against a nuclease-urea extract of HeLa metaphase chromosomes. This antibody stains metaphase chromosomes from a variety of mammalian cultured cell types by indirect immunofluorescence. Antibody 12C7 reacts by western transfer technique with histone H1 in all the cell lines tested. The antibody cross-reacts with H1, and H1(0) in human cells. Proteolytic digestions of H1 suggest that the epitope is localized in the carboxy-terminal domain of the histone H1 molecule. Digestion with trypsin demonstrates that the antibody 12C7 does not react with the globular domain of histone H1. The C-terminal domain of H1 subtypes therefore seems to have a conserved determinant which does exist in H1, H1(0), and probably in H5. This antibody has applications in studying the role of that domain of H1 in processes like chromosome condensation and variations in chromatin structure which influence gene expression.

Antibodies, Monoclonal↗