ABSTRACT
The first centromeric protein identified in any species was CENP-A, a divergent member of the histone H3 family that was recognised by autoantibodies from patients with scleroderma-spectrum disease. It has recently been suggested to rename this protein CenH3. Here, we argue that the original name should be maintained both because it is the basis of a long established nomenclature for centromere proteins and because it avoids confusion due to the presence of canonical histone H3 at centromeres.
Subject(s)
Autoantigens/genetics , Chromosomal Proteins, Non-Histone/genetics , Histones/genetics , Autoantigens/metabolism , Centromere , Centromere Protein A , Chromosomal Proteins, Non-Histone/metabolism , Histones/metabolism , Humans , Kinetochores , Scleroderma, Systemic/genetics , Terminology as TopicABSTRACT
BACKGROUND: Cell biologists face the need to rapidly analyse their proteins of interest in order to gain insight into their function. Often protein purification, cellular localisation and Western blot analyses can be multi-step processes, where protein is lost, activity is destroyed or effective antibodies have not yet been generated. AIM: To develop a method that simplifies the critical protein analytical steps of the laboratory researcher, leading to easy, efficient and rapid protein purification, cellular localisation and quantification. RESULTS: We have tagged the SMC2 subunit of the condensin complex with the Streptavidin-Binding Peptide (SBP), optimising and demonstrating the efficacious use of this tag for performing these protein analytical steps. Based on silver staining, and Western analysis, SBP delivered an outstanding specificity and purity of the condensin complex. We also developed a rapid and highly specific procedure to localise SBP-tagged proteins in cells in a single step procedure thus bypassing the need for using antibodies. Furthermore we have shown that the SBP tag can be used for isolating tagged proteins from chemically cross-linked cell populations for capturing DNA-protein interactions. CONCLUSIONS: The small 38-amino acid synthetic SBP offers the potential to successfully perform all four critical analytical procedures as a single step and should have a general utility for the study of many proteins and protein complexes.
Subject(s)
Adenosine Triphosphatases/metabolism , Carrier Proteins/metabolism , DNA-Binding Proteins/metabolism , Multiprotein Complexes/metabolism , Nuclear Proteins/metabolism , Adenosine Triphosphatases/chemistry , Adenosine Triphosphatases/isolation & purification , Animals , Blotting, Western , Carrier Proteins/chemistry , Carrier Proteins/genetics , Cell Cycle Proteins , Cell Line, Tumor , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/isolation & purification , Fluorescence , Humans , Microscopy, Fluorescence , Multiprotein Complexes/chemistry , Multiprotein Complexes/isolation & purification , Mutation , Nuclear Proteins/chemistry , Nuclear Proteins/genetics , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Reproducibility of Results , Streptavidin/chemistry , Streptavidin/metabolismABSTRACT
The centromere is a complex structure required for equal segregation of newly synthesised sister chromatids at mitosis. One of the significant objectives in centromere research is to determine the complete repertoire of protein components that constitute the kinetochore. Here, we identify a novel centromere protein using a centromere-positive autoimmune serum from a patient with watermelon stomach disease. Western blot and screening of a lambda phage expression library revealed a 60-kDa protein, ZNF397. This protein belongs to the classical Cys(2)His(2) group of the zinc-finger protein superfamily and contains two conserved domains: a leucine-rich SCAN domain and nine Cys(2)His(2) zinc fingers. Bioinformatic analysis shows that ZNF397 is conserved in placental mammals. Stable GFP:ZNF397-expressing human cells show co-localisation of ZNF397 with the constitutive centromere protein CENP-A during interphase and early prophase. Deletion and domain-swap constructs indicate that the SCAN domain is necessary but not sufficient for centromere localisation. Gene-knockout studies in mice using the mouse orthologue (Zfp397) reveal that ZNF397 is a non-essential protein. These properties define ZNF397 as a member of a new class of interphase to early prophase-specific and SCAN domain-containing mammalian centromere protein. The possible role of this protein in transcription at the centromere is discussed.
Subject(s)
Centromere/genetics , Interphase/genetics , Protein Structure, Tertiary/genetics , Transcription Factors/genetics , Zinc Fingers/genetics , Animals , Blotting, Western , Computational Biology , Gastric Antral Vascular Ectasia/immunology , Gene Components , HeLa Cells , Humans , Immune Sera/immunology , Mice , Mice, Knockout , Microscopy, Fluorescence , Species SpecificityABSTRACT
Centromere protein B is a highly conserved constitutive protein found at centromeres. Gene knockout studies in mice have unexpectedly identified Cenpb as a candidate gene involved in uterine function. The present study further explores the role of Cenpb in mice by intermating Cenpb-null mice over several generations. Breeding studies and analysis of uterine tissue indicate that Cenpb-null mice lose reproductive fitness over a number of generations due to a significant reduction in endometrial glands. These results suggest that Cenpb may play an important function in the short- and long-term maintenance of uterine integrity.
Subject(s)
Autoantigens , Chromosomal Proteins, Non-Histone/physiology , DNA-Binding Proteins , Endometrium/metabolism , Reproduction/physiology , Animals , Breeding , Centromere Protein B , Chromosomal Proteins, Non-Histone/analysis , Chromosomal Proteins, Non-Histone/genetics , Endometrium/pathology , Female , Genomics , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Organ Size/genetics , Ovary/pathology , Ovary/transplantationSubject(s)
Aneuploidy , Polymerase Chain Reaction/methods , Prenatal Diagnosis/methods , Female , Humans , Pregnancy , Prospective Studies , Time FactorsABSTRACT
Human neocentromeres are fully functional centromeres that arise naturally in non-centromeric regions devoid of alpha-satellite DNA. We have successfully produced a series of minichromosomes by telomere-associated truncation of a marker chromosome mardel(10) containing a neocentromere. The resulting minichromosomes are either linear or circular in nature, and range in size from approximately 650 kb to 2 Mb. These minichromosomes exhibit full centromeric activity, bind to essential centromere proteins, and are mitotically stable over many generations. They provide a useful system for dissecting the functional domains of complex eukaryotic centromeres and as vectors for therapeutic gene delivery.
