Expression of an Nkx3.1-CRE gene using ROSA26 reporter mice.

The genetic locus of Nkx3.1, an early murine marker of sclerotome and prostate development, was disrupted by a knock in of CRE recombinase via homologous recombination in embryonic stem cells. Cell fate mapping revealed previously unidentified cell lineages expanded from Nkx3.1-expressing cell populations and recapitulated reported Nkx3.1 expression patterns. In lineage trace experiments of E18.5 Nkx3.1-CRE; R26R embryos novel staining was observed in areas of the lungs, portions of the duodenum, and vertebral elements of the skeleton. beta-galactosidase activity measured in Nkx3.1-CRE; R26R and Nkx3.2-CRE; R26R embryos was observed in overlapping regions of the sclerotome but no apparent change in Nkx3.1 expression was seen in the Nkx3.2 mutants by in situ hybridization.

Erythrocyte spectrin's chimeric E2/E3 ubiquitin conjugating/ligating activity.

Spectrin is important for the shape and the physical properties of the red blood cell, such as deformability and resistance to mechanical stress. Previous findings from our laboratory indicated that human erythrocyte alpha-spectrin can facilitate formation of ubiquitin-spectrin adducts and conjugates. Computer analysis revealed domains that contained significant homologies to known consensus catalytic E2 and E3 sequences, and allowed us to develop a model for alpha-spectrin ubiquitin conjugating enzyme (E2) and ubiquitin protein ligase (E3) enzymatic activities. In order to identify the precise E2/E3 site(s), in the present study, a GST-fusion alpha-spectrin (2005-2415) recombinant protein was tested using a cell free in vitro ubiquitination assay. We found that cysteine 2071 and cysteine 2100 are critical for alpha-spectrin (2005-2415) E2/E3 activity. Furthermore, together with testing an additional 13 site-specific mutants, we also demonstrated that both Cys2071 and Cys2100 are capable of transferring ubiquitin from an E1 enzyme to target sites within alpha-spectrin (2005-2415).

Nuclear entry of nonviral vectors.

Nonviral gene delivery is limited to a large extent by multiple extracellular and intracellular barriers. One of the major barriers, especially in nondividing cells, is the nuclear envelope. Once in the cytoplasm, plasmids must make their way into the nucleus in order to be expressed. Numerous studies have demonstrated that transfections work best in dividing populations of cells in which the nuclear envelope disassembles during mitosis, thus largely eliminating the barrier. However, since many of the cells that are targets for gene therapy do not actively undergo cell division during the gene transfer process, the mechanisms of nuclear transport of plasmids in nondividing cells are of critical importance. In this review, we summarize recent studies designed to elucidate the mechanisms of plasmid nuclear import in nondividing cells and discuss approaches to either exploit or circumvent these processes to increase the efficiency of gene transfer and therapy.

Regulation of organ development by the NKX-homeodomain factors: an NKX code.

Mammalian development is a highly coordinated process that involves sequential and time-dependent gene regulation. Deregulation of this process can have functional or morphological consequences, possibly causing lethality or organ dysfunction. Homeotic genes are considered the master regulators of early developmental processes. Of the many homeodomain genes, the NK2 class represents a family of phylogenetically ancient proteins. NK2 homeobox family members are tissue-specific transcription factors distinguished by a common DNA binding structure unique among the homeodomain genes. Increasing evidence indicates that individual Nkx factors are critical regulators of whole organ development. In the sections below, we review the structure, regulation, and expression of the NK2 gene family beginning with their discovery in Drosophila and relating the known features of vertebrate counterparts to the Drosophilaproteins. In particular, we note that each of the vertebrate NK2 proteins are associated with particular genetic anomalies leading to a variety of described disease states. Further, based upon our examination we propose a new paradigm of development based upon the regulatory capacity of the NK2 homeodomain proteins termed the "Nkx Code"

The smooth muscle gamma-actin gene is androgen responsive in prostate epithelia.

Nkx 3.1 is an evolutionarily conserved vertebrate homolog of the Drosophila Nk-3 homeodomain gene bagpipe that is expressed by a variety of cells during early mammalian development and has been shown to be a critical factor for prostate development and function. Previous studies utilizing a heterologous cell transfection strategy from our laboratory identified the smooth muscle gamma-actin (SMGA) gene as a novel molecular target of Nkx 3.1 regulatory activity. In the studies presented here, SMGA gene activity and regulation were evaluated in normal and cancerous prostate epithelial cells. SMGA transcripts were demonstrated in prostate epithelia and SMGA mRNA levels were increased in androgen-responsive LNCaP cancer and normal prostate epithelial cells. SMGA gene transcriptional activity was androgen responsive in these cells and required a segment of the human SMGA promoter containing NKE and SRF (serum response factor) binding elements. This region of the human SMGA proximal promoter is well conserved across species and is synergistically activated by coexpression of Nkx 3.1 and SRF in heterologous CV-1 cells. SMGA transcription was not responsive to steroid in PC-3 prostate epithelial cancer cells, which do not express Nkx 3.1. However, SMGA transcription was influenced by expression of androgen receptor in these cells, a situation that allows the androgen-dependent expression of Nkx 3.1. Furthermore, SMGA gene activity was influenced by direct Nkx 3.1 expression in the PC-3 cells. Thus, SMGA gene activity in prostate epithelia is due, in part, to the androgen-dependent expression of Nkx 3.1. As such, our studies provide the initial description of Nkx 3.1 target gene regulatory activity in the prostate.

Erythrocyte spectrin is an E2 ubiquitin conjugating enzyme.

The involvement of red blood cell spectrin in the ubiquitination process was studied. Spectrin was found to form two ubiquitin-associated derivatives, a DTT-sensitive ubiquitin adduct and a DTT-insensitive conjugate, characteristic intermediate and final products of the ubiquitination reaction cascade. In addition to spectrin and ubiquitin, ubiquitin-activating enzyme (E1) and ATP were necessary and sufficient to form both the spectrin-ubiquitin adduct and conjugate. No exogenous ubiquitin-conjugating (E2) or ligase (E3) activities were required, suggesting that erythrocyte spectrin is an E2 ubiquitin-conjugating enzyme able to target itself. Both ubiquitin adduct and conjugate were linked to the alpha subunit of spectrin, suggesting that the ubiquitin-conjugating (UBC) domain and its target regions reside on the same subunit.

Molecular characterization and distribution of the opioid growth factor receptor (OGFr) in mouse.

The native opioid growth factor (OGF), [Met(5)]-enkephalin, is a tonic inhibitory peptide that modulates cell proliferation and tissue organization during development, cancer, cellular renewal, wound healing, and angiogenesis. OGF action is mediated by a receptor mechanism. The receptor for OGF, OGFr, has been cloned and sequenced in humans and rats. Using primers based on the rat OGFr cDNA, and a mouse embryo expressed sequence tag, the full-length 2.1 kb mouse OGFr cDNA was sequenced. The open reading frame was found to encode a protein of 634 amino acids, and 14 imperfect repeats of 9 amino acids each were a prominent feature. The molecular weight of OGFr was calculated as 70679, and the isoelectric point was 4.5. Northern blot analysis revealed a 2.1 kb OGFr mRNA transcript in adult mouse brain, heart, lung, liver, kidney, and triceps surae muscle. The amino acids for mouse and rat OGFr were 93% similar and 91% identical, but the mouse and human shared only a 70% similarity and a 58% identity. These results emphasize the molecular validity of OGFr, and explain the interaction of OGF with respect to normal and abnormal growth in mouse cells and tissues.

The domain of brain beta-spectrin responsible for synaptic vesicle association is essential for synaptic transmission.

We have examined the interaction between synapsin I, the major phosphoprotein on the membrane of small synaptic vesicles, and brain spectrin. Using recombinant peptides we have localized the synapsin I attachment site upon the beta-spectrin isoform betaSpIISigmaI to a region of 25 amino acids, residues 211 through 235. This segment is adjacent to the actin binding domain and is within the region of the betaSpIISigmaI that we previously predicted as a candidate synapsin I binding domain based upon sequence homology. We used differential centrifugation techniques to quantitatively assess the interaction of spectrin with synaptic vesicles. Using this assay, high affinity saturable binding of recombinant betaSpIISigmaI proteins was observed with synaptic vesicles. Binding was only observed when the 25 amino acid synapsin I binding site was included on the recombinant peptides. Further, we demonstrate that antibodies directed against 15 amino acids of the synapsin I binding domain specifically blocked synaptic transmission in cultured hippocampal neurons. Thus, the synapsin I attachment site on betaSpIISigmaI spectrin comprises a approximately 25 amino acid segment of the molecule and interaction of these two proteins is an essential step for the process of neurotransmission.

The smooth muscle gamma-actin gene promoter is a molecular target for the mouse bagpipe homologue, mNkx3-1, and serum response factor.

An evolutionarily conserved vertebrate homologue of the Drosophila NK-3 homeodomain gene bagpipe, Nkx3-1, is expressed in vascular and visceral mesoderm-derived muscle tissues and may influence smooth muscle cell differentiation. Nkx3-1 was evaluated for mediating smooth muscle gamma-actin (SMGA) gene activity, a specific marker of smooth muscle differentiation. Expression of mNkx3-1 in heterologous CV-1 fibroblasts was unable to elicit SMGA promoter activity but required the coexpression of serum response factor (SRF) to activate robust SMGA transcription. A novel complex element containing a juxtaposed Nkx-binding site (NKE) and an SRF-binding element (SRE) in the proximal promoter region was found to be necessary for the Nkx3-1/SRF coactivation of SMGA transcription. Furthermore, Nkx3-1 and SRF associate through protein-protein interactions and the homeodomain region of Nkx3-1 facilitated SRF binding to the complex NKE.SRE. Mutagenesis of Nkx3-1 revealed an inhibitory domain within its C-terminal segment. In addition, mNkx3-1/SRF cooperative activity required an intact Nkx3-1 homeodomain along with the MADS box of SRF, which contains DNA binding and dimerization structural domains, and the contiguous C-terminal SRF activation domain. Thus, SMGA is a novel target for Nkx3-1, and the activity of Nkx3-1 on the SMGA promoter is dependent upon SRF.

Cell-specific nuclear import of plasmid DNA.

One factor limiting the success of non-viral gene therapy vectors is the relative inability to target genes specifically to a desired cell type. To address this limitation, we have begun to develop cell-specific vectors whose specificity is at the level of the nuclear import of the plasmid DNA. We have recently shown that nuclear import of plasmid DNA is a sequence-specific event, requiring the SV40 enhancer, a region known to bind to a number of general transcription factors (Dean DA, Exp Cell Res 1997; 230: 293). From these studies we developed a model whereby transcription factor(s) bind to the DNA in the cytoplasm to create a protein-DNA complex that can enter the nucleus using the protein import machinery. Our model predicts that by using DNA elements containing binding sites for transcription factors expressed in unique cell types, we should be able to create plasmids that target to the nucleus in a cell-specific manner. Using the promoter from the smooth muscle gamma actin (SMGA) gene whose expression is limited to smooth muscle cells, we have created a series of reporter plasmids that are expressed selectively in smooth muscle cells. Moreover, when injected into the cytoplasm, plasmids containing portions of the SMGA promoter localize to the nucleus of smooth muscle cells, but remain cytoplasmic in fibroblasts and CV1 cells. In contrast, a similar plasmid carrying the SV40 enhancer is transported into the nuclei of all cell types tested. Nuclear import of the SMGA promoter-containing plasmids could be achieved when the smooth muscle specific transcription factor SRF was expressed in stably transfected CV1 cells, supporting our model for the nuclear import of plasmids. Finally, these nuclear targeting sequences were also able to promote increased gene expression in liposome- and polycation-transfected non-dividing cells in a cell-specific manner, similar to their nuclear import activity. These results provide proof of principle for the development of cell-specific non-viral vectors for any desired cell type.

Cell-specific transcription of the smooth muscle gamma-actin gene requires both positive- and negative-acting cis elements.

We have characterized the function of putative regulatory sequences upon the smooth muscle transcription of the SMGA gene, using promoter deletion analyses. We demonstrate that the SMGA promoter contains four domains: a basal promoter (-1 to -100), a smooth muscle specifier sequence (-100 to -400), a negative regulator (-400 to -1000), and a smooth muscle-specific modulator (-1000 to -2000). The basal or core promoter supports equivalent transcription in both smooth and skeletal muscle cells. Addition of sequences containing a CArG motif juxtaposed to an E-box element stimulates smooth muscle transcription by five- to sixfold compared to skeletal muscle. This smooth muscle-specific segment is maintained for about 200 bp, after which is a segment of DNA that appears to inhibit the transcriptional capacity of the SMGA promoter in smooth muscle cells. Within the boundary between the smooth muscle specifier and negative regulatory sequences (-400 to -500) are three E-box elements. The smooth muscle modulator domain contains two CArG elements and multiple E-boxes. When added to the SMGA promoter it causes an additional three- to fivefold increase in smooth muscle-specific transcription over that stimulated by the smooth muscle specifier domain. Thus, our studies show that the appropriate cell-specific transcription of the SMGA gene involves complex interactions directed by multiple cis-acting elements. Moreover, our characterization of a cell culture system employing embryonic gizzard smooth muscle cells lays the foundation for further molecular analyses of factors that regulate or control SMGA and other smooth muscle genes during differentiation.

The effect of human leukocyte antigen disparity on cyclosporine neurotoxicity after allogeneic bone marrow transplantation.

PURPOSE: We examined the relationship between human leukocyte antigen (HLA) matching and the development of cyclosporine (CyA) neurotoxicity in patients undergoing allogeneic bone marrow transplantation, and determined the frequency and imaging characteristics of CyA neurotoxicity in these patients. METHODS: Records of 87 patients who underwent allogeneic bone marrow transplantation were reviewed. Eight patients who presented with visual disturbance and/or seizures and had MR imaging within 24 hours were identified. Transplant donor relatedness was examined, and patients' imaging studies were reviewed. Clinical parameters, including blood pressure, CyA, creatinine, and magnesium levels, and the presence of graft-versus-host disease were reviewed. RESULTS: CyA neurotoxicity was seen more frequently in HLA-mismatched and unrelated donor transplants. The frequency of CyA neurotoxicity was 4% for patients with a 5/6 or 6/6 HLA match, 13% for matched unrelated donor transplants, and 50% for haplotypic 3/6 or 4/6 transplants. Patients with matched unrelated donor transplants and haplotypic transplants presented earlier in the posttransplant time course and had decreased survival time relative to patients with HLA-matched transplants. Imaging abnormalities most commonly affected the occipital lobes and the posterior cerebral hemispheres; both cortical and white matter involvement was identifiable as T1 hypointense and T2 hyperintense signal with associated gyral swelling and sulcal effacement on the initial MR studies. Hypodensity in the affected areas was noted on CT scans. Contrast enhancement was seen in HLA-mismatched and unrelated transplants only. Follow-up imaging showed interval decreases in subcortical edema; however, residual signal abnormality, primarily affecting the cortex, was present in all cases and seen best on proton density-weighted MR images. CONCLUSION: The frequency of severe CyA neurotoxicity increases with increasing HLA disparity, suggesting that immune factors may play a role. CyA neurotoxicity appears to represent a spectrum of disease processes. Disruption of the blood-brain barrier as well as hypoxic or vasculitic cortical injury resulting in MR-detectable cortical signal abnormalities may occur in severe cases.

Localization of autoepitopes on the PCM-1 autoantigen using scleroderma sera with autoantibodies against the centrosome.

Characterization of epitope domains of autoantigens is important for deducing the cellular functions of autoantigens and may be important for understanding the autoimmune response. In the reported studies, epitope analysis of the centrosome autoantigen PCM-1 was performed. For these investigations, portion of the PCM-1 cDNA were subcloned into the pMAL expression plasmid, fusion proteins were induced, and aliquots of the extracts were probed by immunoblot analysis using two human autoimmune anticentrosome autoantisera. Immunoblotting identified three individual autoepitopes of 26-40 amino acid residues, amino acids 506-545, 1434-1465, and 1661-1686, within the PCM-1 protein. ELISA assays using non-denatured proteins did not identity any additional autoepitopes in the remainder of the PCM-1 molecule. To analyze the identified autoepitopes further, synthetic peptides were generated that covered each of the three autoepitopes and the synthetic peptides then were probed using the scleroderma sera. Peptides that covered the antigenic regions from amino acids 506-545 and 1434-1465 failed to react with the anticentrosome autoantisera suggesting that overall protein conformation may be important for the formation of those two autoepitopes. Peptides derived from the sequence of the third autoepitope were recognized by autoantibodies present in the anticentrosome autoantisera allowing the identification of the tripeptide KDC as the autoepitope in this region of the PCM-1 molecule. These studies lay the foundation for future investigations of the autoimmune response in scleroderma patients that are producing anticentrosome autoantibodies and should allow an investigation of the cellular role of the PCM-1 protein.

The developmentally regulated expression of serum response factor plays a key role in the control of smooth muscle-specific genes.

Serum response factor (SRF) is a MADS box transcription factor that has been shown to be important in the regulation of a variety of muscle-specific genes. We have previously shown SRF to be a major component of multiple cis/trans interactions found along the smooth muscle gamma-actin (SMGA) promoter. In the studies reported here, we have further characterized the role of SRF in the regulation of the SMGA gene in the developing gizzard. EMSA analyses, using nuclear extracts derived from gizzards at various stages in development, showed that the SRF-containing complexes were not present early in gizzard smooth muscle development, but appeared as development progressed. We observed an increase in SRF protein and mRNA levels during gizzard development by Western and Northern blot analyses, with a large increase just preceding an increase in SMGA expression. Thus, changes in SRF DNA-binding activity were paralleled with increased SRF gene expression. Immunohistochemical analyses demonstrated a correspondence of SRF and SMGA expression in differentiating visceral smooth muscle cells (SMCs) during gizzard tissue development. This correspondence of SRF and SMGA expression was also observed in cultured smooth muscle mesenchyme induced to express differentiated gene products in vitro. In gene transfer experiments with SMGA promoter-luciferase reporter gene constructs we observed four- to fivefold stronger SMGA promoter activity in differentiated SMCs relative to replicating visceral smooth muscle cells. Further, we demonstrate the ability of a dominant negative SRF mutant protein to specifically inhibit transcription of the SMGA promoter in visceral smooth muscle, directly linking SRF with the control of SMGA gene expression. Taken together, these data suggest that SRF plays a prominent role in the developmental regulation of the SMGA gene.

S100A1 and S100B expression and target proteins in type I diabetes.

Calcium receptor proteins are an essential link between hormones that alter intracellular calcium levels and the generation of cellular responses. However, there is no information available regarding the role of calcium receptor proteins, in particular the S100 family, in insulin action and/or diabetes. This study examines the effects of streptozotocin-induced type I diabetes on the expression of the individual S100A1 and S100B isoforms as well as their binding proteins. Diabetes did not increase (or initiate) S100B expression in any non-S100B-expressing tissue (skeletal muscle, heart, kidney, liver, spleen, and pancreas). In all S100B-expressing tissues examined (brain, white fat, and testes), S100B protein levels increased approximately 2-fold while steady state S100B messenger RNA (mRNA) levels decreased. S100A1-expressing tissues exhibited increased (kidney and lung), decreased (skeletal muscle), and unchanged (brain and heart) S100A1 protein levels. While noncoordinate changes in S100A1 protein and steady state mRNA levels were observed in heart, other S100A1-expressing tissues (brain, slow twitch skeletal muscle, and kidney) exhibited coordinate changes in S100A1 protein and steady state mRNA levels. Altogether, these results suggest that the effects of diabetes on S100 expression are isoform as well as tissue-specific. Gel overlay analysis of the S100-binding protein profile revealed both increases and decreases in binding proteins in all tissues examined. In summary, changes in the expression of S100A1, S100B, and S100-binding proteins occur in type I diabetes and represent important molecular events in the effects of insulin/insulin insufficiency on cell function.

Dissociation of centrosome replication events from cycles of DNA synthesis and mitotic division in hydroxyurea-arrested Chinese hamster ovary cells.

Relatively little is known about the mechanisms used by somatic cells to regulate the replication of the centrosome complex. Centrosome doubling was studied in CHO cells by electron microscopy and immunofluorescence microscopy using human autoimmune anticentrosome antiserum, and by Northern blotting using the cDNA encoding portion of the centrosome autoantigen pericentriolar material (PCM)-1. Centrosome doubling could be dissociated from cycles of DNA synthesis and mitotic division by arresting cells at the G1/S boundary of the cell cycle using either hydroxyurea or aphidicolin. Immunofluorescence micros-copy using SPJ human autoimmune anticentrosome antiserum demonstrated that arrested cells were able to undergo numerous rounds of centrosome replication in the absence of cycles of DNA synthesis and mitosis. Northern blot analysis demonstrated that the synthesis and degradation of the mRNA encoding PCM-1 occurred in a cell cycle-dependent fashion in CHO cells with peak levels of PCM-1 mRNA being present in G1 and S phase cells before mRNA amounts dropped to undetectable levels in G2 and M phases. Conversely, cells arrested at the G1/S boundary of the cell cycle maintained PCM-1 mRNA at artificially elevated levels, providing a possible molecular mechanism for explaining the multiple rounds of centrosome replication that occurred in CHO cells during prolonged hydroxyurea-induced arrest. The capacity to replicate centrosomes could be abolished in hydroxyurea-arrested CHO cells by culturing the cells in dialyzed serum. However, the ability to replicate centrosomes and to synthesize PCM-1 mRNA could be re-initiated by adding EGF to the dialyzed serum. This experimental system should be useful for investigating the positive and negative molecular mechanisms used by somatic cells to regulate the replication of centrosomes and for studying and the methods used by somatic cells for coordinating centrosome duplication with other cell cycle progression events.

Brain spectrin: of mice and men.

This article reviews our current knowledge of the structure of alpha spectrins and beta spectrins in the brain, as well as their location and expression within neural tissue. We discuss the known protein interactions of brain spectrin isoforms, and then describe results that suggest an important role for spectrin (alpha SpII sigma 1/beta SpII sigma 1) in the Ca(2+)-regulated release of neurotransmitters. Evidence that supports a role for spectrin in the docking of synaptic vesicles to the presynaptic plasma membrane and as a Ca2+ sensor protein that unclamps the fusion machinery is described, along with the Casting the Line model, which summarizes the information. We finish with a discussion of the value of spectrin and ankyrin-deficient mouse models in deciphering spectrin function in neural tissue.

Autoepitope mapping of the centrosome autoantigen PCM-1 using scleroderma sera with anticentrosome autoantibodies.

We previously characterized a scleroderma serum (serum 1) containing autoantibodies against centrosome autoantigens that have been named PCM-1, PCM-2 and PCM-3. In this study, we analyzed another scleroderma serum (serum 2) reactive with centrosome autoantigens of identical molecular weights to those recognized by serum 1. To further analyze the autoepitope domains in PCM-1 recognized by the autoantibodies present in scleroderma sera, cDNAs encoding different portions of the PCM-1 autoantigen were expressed in bacteria as fusion proteins. The immunoreactivity of the fusion proteins to the scleroderma sera was assayed by immunoblot analysis. Two regions containing autoepitope domains reactive with both sera were identified in the PCM-1 molecule. One is between amino acids 312-706 of the PCM-1 autoantigen, and the other is localized between amino acids 1,433-1,787, indicating that the immune response is oligoclonal. The results are important to clarify the mechanism of induction of anticentrosome autoantibodies. The potential diagnostic and prognostic significance of the autoantibodies for subgroups of scleroderma is discussed.

Brain alpha erythroid spectrin: identification, compartmentalization, and beta spectrin associations.

Using isoform and subunit specific antibodies we have determined the presence, localization, and beta spectrin associations of alpha erythroid spectrin, alpha SpI sigma*, as well as alpha non-erythroid spectrin, alpha SpII sigma 1, in mouse brain. Peptide specific antibodies against unique sequences within the beta SpII sigma 1, non-erythroid beta spectrin isoform, and within beta SpI sigma 1, erythrocyte beta spectrin isoform were used to compare the immunolocalization of beta spectrin subunit isoforms with that of alpha spectrin subunit isoforms and to immunoprecipitate spectrin tetramers in order to identify the subunit components by immunoblot analysis. The specificity and sensitivity of antibodies for isoform specific alpha and beta subunits was determined by immunodot and immunoblot methods. Immunohistochemical analyses indicated that beta SpI sigma 2 is located in neuronal somata and dendrites in mouse cerebellum. beta SpII sigma 1 is located in the medullary layer, chiefly composed of axonal tracts. Parallel immunohistochemical analysis with antibodies for the alpha and beta spectrin isoforms revealed that antibodies specific for the alpha subunit of erythrocyte spectrin (alpha SpI sigma 1) localized antigen to the somata and dendrites of cerebellar granule cell neurons, a pattern similar to that for the localization of the erythroid beta subunit (beta SpI sigma 2). In contrast antibodies specific for the non-erythroid alpha subunit (alpha SpII sigma 1) localized antigen to axons in the cerebellum corresponding to the pattern for the non-erythroid beta subunit (beta SpII sigma 1). The distinct localization of antigens by antisera which recognize either the alpha subunit of red blood cell spectrin or the alpha subunit of non-erythroid brain spectrin, together with the correspondence of their localization with appropriate beta subunits, clearly indicate that brain contains at least two species of spectrin each with distinct alpha and beta subunits. Immunoprecipitation experiments of cerebellar extracts using beta spectrin peptide specific antibodies followed by immunoblotting analysis confirmed the association of an erythroid alpha subunit isoform with a beta erythroid subunit isoform, as well as the association of non-erythroid alpha and beta subunits. In addition the immunoblot analysis of the immunoprecipitated material suggested there are minor populations of various hybrid tetramers in brain consisting of mixed erythroid and non-erythroid subunits. In summary these data collectively demonstrate that in mouse brain there are at least two alpha spectrin subunits, one erythroid alpha SpI sigma* and one non-erythroid alpha SpII sigma 1; these associate with an erythroid beta SpI sigma 1, and a non-erythroid beta SpII sigma 1 in the cerebellum of mouse.(ABSTRACT TRUNCATED AT 400 WORDS)

PCM-1, A 228-kD centrosome autoantigen with a distinct cell cycle distribution.

We report the identification and primary sequence of PCM-1, a 228-kD centrosomal protein that exhibits a distinct cell cycle-dependent association with the centrosome complex. Immunofluorescence microscopy using antibodies against recombinant PCM-1 demonstrated that PCM-1 is tightly associated with the centrosome complex through G1, S, and a portion of G2. However, late in G2, as cells prepare for mitosis, PCM-1 dissociates from the centrosome and then remains dispersed throughout the cell during mitosis before re-associating with the centrosomes in the G1 phase progeny cells. These results demonstrate that the pericentriolar material is a dynamic substance whose composition can fluctuate during the cell cycle.