Preferential affinity of high molecular weight high mobility group non-histone chromatin proteins for single-stranded DNA.

We have subjected proteins dissociated from chicken erythrocyte or calf thymus chromatin by 0.35 M NaCl to sequential chromatography on columns containing immobilized double-stranded DNA and single-stranded DNA. At 0.2 M NaCl, 1 mM Tris . Cl (pH 7.5), the high molecular weight, high mobility group proteins (HMG-1, HMG-2, and HMG-E), were not retained by double-stranded DNA columns, but were retained by single-stranded DNA columns. Thus, in that solvent, those proteins exhibit selective affinity for single-stranded DNA. This suggests that the functions of the high molecular weight, high mobility group proteins might involve destabilizing the DNA double helix by virtue of their preferential affinity for single-stranded DNA.     

Nonhistone chromatin proteins HMG-14 and HMG-17 bind preferentially to single-stranded DNA.

Proteins extracted from chicken erythrocyte chromatin with 0.35 M NaCl were subjected to sequential chromatography on columns containing immobilized double-stranded and single-stranded DNA's. Two-dimensional electrophoresis of protein fractions revealed that HMG-14 and HMG-17 are among the proteins that are retained by the single-stranded DNA column in 0.2 M NaCl/l mM Tris-Cl (pH 7.5) after having failed to be retained by the double-stranded column under the same conditions. That suggests that those two proteins possess preferential affinity for single-stranded DNA. Further evidence for that was provided by chromatography of purified HMG-14 and of purified HMG-17 on single-stranded and double-stranded DNA columns. We discuss the possible relevance of our results to suggested functions of HMG-14 and HMG-17.     

The chromatin repeat length of brain cortex and cerebellar neurons changes concomitant with terminal differentiation.

Chromatin repeat lengths in neuronal, glial, and liver nuclei of the rat were determined by micrococcal nuclease digestion followed by gel electrophoresis. The repeat length of cortex neurons decreased from 200 base pairs (bp) before birth to 170 bp at 14 days and all subsequent stages. Administration of [3H]thymidine to pregnant rats during the period of fetal neurogenesis allowed neurons differing in their time of origin to be labeled individually. This revealed that the shortening of the chromatin repeat length affected only neurons generated early during development, i.e., between gestational days 13/14 and 18/19, whereas neurons continuing to proliferate beyond gestational day 19 and up to birth (day 22) did not undergo shortening of their repeat length. In contrast to the cortex neurons, cerebellar neurons (granule cells) underwent lengthening of the repeat length from 165 bp at fetal and early post-natal stages (up to day 4) to 218 bp after day 30. Thus, in both cortex and cerebellar neurons the changes occurred temporally coincident with major developmental processes. No changes were detected in liver nuclei during the same period. Non-astrocytic glia cells of the adult cortex had 200 bp repeats.     

Mouse embryos and chimera cloned from neural cells in the postnatal cerebral cortex

Cloning of mice has been achieved by transferring nuclei of various types of somatic cell nuclei into enucleated oocytes. However, all attempts to produce live cloned offspring using the nuclei of neurons from adult cerebral cortex have failed. Previously we obtained cloned mice using the nuclei of neural cells collected from fetal cerebral cortex. Here, we attempted to generate cloned mice using differentiated neurons from the cerebral cortex of postnatal (day 0-4) mice. Although we were unable to obtain live cloned pups, many fetuses reached day 10.5 days of development. These fetuses showed various abnormalities such as spherical omission of the neuroepithelium, collapsed lumen of neural tube, and aberrant expressions of marker proteins of neurons. We produced chimeric mice in which some hair cells and kidney cells were originated from differentiated neurons. In chimeric fetuses, LacZ-positive donor cells were in all three germ cell layers. However, chimeras with large contribution of donor-derived cells were not obtained. These results indicate that nuclei of differentiated neurons have lost their developmental totipotency. In other words, the conventional nuclear transfer technique does not allow nuclei of differentiated neurons to undergo complete genomic reprogramming required for normal embryonic development.     

The lef-3 gene of Autographa californica nuclear polyhedrosis virus encodes a single-stranded DNA-binding protein.

The Autographa californica nuclear polyhedrosis virus (AcNPV) replicates in the nuclei of infected cells and encodes several proteins required for viral DNA replication. As a first step in the functional characterization of viral replication proteins, we purified a single-stranded DNA-binding protein (SSB) from AcNPV-infected insect cells. Nuclear extracts were chromatographed on single-stranded DNA agarose columns. An abundant protein with an apparent molecular weight of 43,000 was eluted from the columns at 0.9 to 1.0 M NaCl. This protein was not evident in extracts prepared from control cells, suggesting that the SSB was encoded by the virus. SSB bound to single-stranded DNA in solution, and binding was nonspecific with respect to base sequence, as single-stranded vector DNA competed as efficiently as single-stranded DNA containing the AcNPV origin of DNA replication. Competition binding experiments indicated that SSB showed a preference for single-stranded DNA over double-stranded DNA. To determine whether SSB was encoded by the lef-3 gene of AcNPV, the lef-3 open reading frame was cloned under the control of the bacteriophage T7 promoter. Immunochemical analyses indicated that LEF-3 produced in bacteria or in rabbit reticulocyte lysates specifically reacted with antiserum produced by immunization with purified SSB. Immunoblot analyses of infected cell extracts revealed that SSB/LEF-3 was detected by 4 h postinfection and accumulated through 48 h postinfection.     

Purification from cultured hepatoma cells of two nonhistone chromatin proteins with preferential affinity for single-stranded DNA: apparent analogy with calf thymus HMG proteins.

Sequential chromatography on double-stranded DNA and single-stranded DNA columns selects two proteins with marked preference for single-stranded DNA from the complex set of proteins that is released by NaCl from chromatin of cultured hepatoma cells. By a number of criteria, these two proteins appear to be analogous to the calf thymus chromatin proteins HMG-1 and HMG-2.     

Deoxyribonucleic acid-binding proteins in vegetative Bacillus subtilis: alterations caused by stage O sporulation mutations.

Deoxyribonucleic acid (DNA)-binding proteins have been compared between wild-type Bacillus subtilis and five sporulation mutants blocked at different stage O loci. Extracts from exponentially growing cells have been fractionated for proteins binding to single-stranded calf thymus DNA-cellulose and double-stranded B. subtilis DNA-cellulose. In nutrient broth, stage O mutations cause an accumulation of proteins with affinity for double-stranded DNA. Suppression of the mutation with extragenic suppressors relieves the accumulation. In minimal glucose medium, the stage O mutations also cause accumulation of proteins with affinity for double-stranded DNA, but the species accumulated are different from those of nutrient broth-grown cells. In neither case did stage O mutations affect proteins with affinity for single-stranded DNA. The results suggest that the products of stage O loci are functional and operative during vegetative growth.     

Solubilization of the Epstein-Barr virus-determined nuclear antigen and its characterization as a DNA-binding protein.

The Eptstein-Barr virus (EBV)-determined nuclear antigen (EBNA) was solubilized from isolate nuclei of two EBV-transformed cell lines- Raji and AW-Ramos, by high-salt treatment. Its DNA-binding properties were studied by DNA-cellulose chromatography and a 51Cr release complement fixation assay. EBNA binds to both double-stranded and single-stranded calf thymus DNA, showing a higher affinity to double-stranded DNA. There was no detectable difference in the DNA binding of EBNA prepared from Raji and AW-Ramos cells.     

Histones and DNA increase synchronously in neurons during early postnatal development of the rat forebrain cortex

Summary Cytophotometric measurements reveal that a temporally coordinate accumulation of histones and DNA occurs in rat cortex neurons between the last gestational day and the end of the third postnatal week. A rapid rise of both constituents around birth is followed by a more protracted synthesis. This leads to adult histone and DNA levels of approximately 3.3 arbitrary units as compared with 2 arbitrary units found in neuronal precursor cells at all foetal stages and in reference diploid nuclei from glia and liver. A secondary DNA increase in the fourth postnatal week, previously suspected on the basis of cytofluorometric measurements using the Schifftype stain BAO (bis-[4-aminophenyl]-1,3,4-oxadiazole), is not substantiated by the present work. This derives from the finding that alternative cytophotometric DNA measurements (ultraviolet absorption scanning) and autoradiographs of neuronal nuclei following repeated injections of [³H]thymidine into the lateral ventricles during the relevant period give no evidence of a further DNA increase after the third postnatal week. Neither does the accumulation of histones (measured by sulfaflavine cytofluorometry) continue beyond day 21. This leads us to conclude that DNA and histone syntheses cease at the end of the third week. Electrophoretic analyses of the histones show that the relative histone composition changes only slightly during neuronal development. Apart from an increase in the ratio of the histones H1^o:H1 around birth no developmental alterations in histone composition are detectable.     

Proliferation of piriform neurons in the rat cerebellar cortex in the pre- and postnatal periods of development]

The method of histoautoradiography with the use of H3-thymidine was applied to the study; there were established the periods of appearance in the cerebellar anlage of albino rats of neuroblasts differentiated into the piriform neurones of the cerebellar cortex; dynamics of the proliferative activity of these cellular elements in the course of the pre- and postnatal periods of development of the experimental animals was investigated. On the basis of the material obtained a conclusion was drawn that the last cell divisions (the result of cell differentiation were Purkinje's cells of the cerebellar cortex) stopped by the 13th--15th day of the embryonic development. No incorporation of the labeled precursor into the DNA of the nuclei of the differentiating piriform neurons occurred later.     

Increase of the DNA strand assimilation activity of recA protein by removal of the C terminus and structure-function studies of the resulting protein fragment

A proteolytic fragment of recA protein, missing about 15% of the protein at the C terminus, was found to promote assimilation of homologous single-stranded DNA into duplex DNA more efficiently than intact recA protein. This difference was not found if Escherichia coli single-stranded DNA binding protein was present. The ATPase activity of both intact recA protein and the fragment was identical. The difference in strand assimilation activity cannot be due to differences in single-stranded DNA affinity, since both the fragment and intact proteins bind to single-stranded DNA with nearly identical affinities. However, the fragment was found to bind double-stranded DNA more tightly and to aggregate more extensively than recA protein; both of these properties may be important in strand assimilation. Aggregation of the fragment was extensive in the presence of duplex DNA under the same condition where recA protein did not aggregate. The double-stranded DNA binding of both recA protein and the fragment responds to nucleotide cofactors in the same manner as single-stranded DNA binding, i.e. ADP weakens and ATP gamma S strengthens the association. The missing C-terminal region of recA protein includes a very acidic region that is homologous to other single-stranded DNA binding proteins and which has been implicated in DNA binding modulation. This C-terminal region may serve a similar function in recA protein, possibly inhibiting double-stranded DNA invasion. The possible role of the enhanced double-stranded DNA affinity of the fragment protein in the mechanism of strand assimilation is discussed.     

Developmental Expression of HNK-1-Reactive Antigens in the Rat Cerebellum and Localization of Sulfoglucuronyl Glycolipids in Molecular Layer and Deep Cerebellar Nuclei

Monoclonal antibody HNK-1-reactive carbohydrate epitope is expressed on proteins, proteoglycans, and sulfoglucuronyl glycolipids (SGGLs). The developmental expression of these HNK-1-reactive antigens was studied in rat cerebellum. The expression of sulfoglucuronyl lacto-N-neotetraosylceramide (SGGL-1) was biphasic with an initial maximum at postnatal day one (PD 1), followed by a second rise in the level at PD 20. The level of sulfoglucuronyl lacto-N-norhexaosyl ceramide (SGGL-2) in cerebellum was low until PD 15 and then increased to a plateau at PD 20. The levels of SGGLs increased during postnatal development of the cerebellum, contrary to their diminishing expression in the cerebral cortex. The expression of HNK-1-reactive glycoproteins decreased with development of the rat cerebellum from PD 1. Several HNK-1-reactive glycoproteins with apparent molecular masses between 150 and 325 kDa were visualized between PD 1 and PD 10. However, beyond PD 10, only two HNK-1-reactive bands at 160 and 180 kDa remained. The latter appeared to be neural cell adhesion molecule, N-CAM-180. A diffuse HNK-1-reactive band seen at the top of polyacrylamide electrophoretic gels was due mostly to proteoglycans. This band increased in its reactivity to HNK-1 between PD 15 and PD 25 and then decreased in the adult cerebellum. The lipid antigens were shown by two complementary methodologies to be localized primarily in the molecular layer and deep cerebellar nuclei as opposed to the granular layer and white matter. A fixation procedure which eliminates HNK-1-reactive epitope on glycoproteins and proteoglycans, but does not affect glycolipids, allowed selective immunoreactivity in the molecular layer and deep cerebellar nuclei.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunolocalization of laminin-alpha1-like antigens around synapses in mouse cerebellar perineuronal nets

The hypothesis that extracellular matrix components may be related to neuronal development in the mouse cerebellar cortex was verified with immunohistochemistry by using an antibody against laminin-alpha1, a major extracellular matrix protein in various tissues. A commercially available polyclonal antibody, raised against the carboxyl-terminal 20-amino acid peptide of laminin-alpha1 was used. Some positive immunoreaction products were localized around large GABAergic interneurons in granular layers and others were around neurons in deep cerebellar nuclei. At the electron microscope level, diaminobenzidine immunoreaction products were localized around presynaptic boutons and in intercellular matrices around interneurons. Such immunoreaction products could be detected at postnatal day 20, when most of cerebellar synapses are assumed to be established. It has been known that a special feature of extracellular matrix, termed perineuronal nets, exists around specific subpopulation of neurons. In the mouse cerebellum, the present findings suggest that laminin itself or laminin-like-antigens exists in the perineuronal nets in relation to inhibitory neuron synapses.     

Immunolocalization of laminin-α1-like antigens around synapses in mouse cerebellar perineuronal nets

The hypothesis that extracellular matrix components may be related to neuronal development in the mouse cerebellar cortex was verified with immunohistochemistry by using an antibody against laminin-α1, a major extracellular matrix protein in various tissues. A commercially available polyclonal antibody, raised against the carboxyl-terminal 20-amino acid peptide of laminin-α1 was used. Some positive immunoreaction products were localized around large GABAergic interneurons in granular layers and others were around neurons in deep cerebellar nuclei. At the electron microscope level, diaminobenzidine immunoreaction products were localized around presynaptic boutons and in intercellular matrices around interneurons. Such immunoreaction products could be detected at postnatal day 20, when most of cerebellar synapses are assumed to be established. It has been known that a special feature of extracellular matrix, termed perineuronal nets, exists around specific subpopulation of neurons. In the mouse cerebellum, the present findings suggest that laminin itself or laminin-like-antigens exists in the perineuronal nets in relation to inhibitory neuron synapses.     

Hyaluronan is organized into fiber-like structures along migratory pathways in the developing mouse cerebellum.

Hyaluronan is a free glycosaminoglycan which is abundant in the extracellular matrix of the developing brain. Although not covalently linked to any protein it can act as a backbone molecule forming aggregates with chondroitin sulfate proteoglycans of the lectican family and link proteins. Using neurocan-GFP as a direct histochemical probe we analyzed the distribution and organization of hyaluronan in the developing mouse cerebellum, and related its fine structure to cell types of specified developmental stages. We observed a high affinity of this probe to fiber-like structures in the prospective white matter which are preferentially oriented parallel to the cerebellar cortex during postnatal development suggesting a specially organized form of hyaluronan. In other layers of the cerebellar cortex, the hyaluronan organization seemed to be more diffuse. During the second postnatal week, the overall staining intensity of hyaluronan in the white matter declined but fiber-like structures were still present at the adult stage. This type of hyaluronan organization is different from perineuronal nets e.g. found in deep cerebellar nuclei. Double staining experiments with cell type specific markers indicated that these fiber-like structures are predominantly situated in regions where motile cells such as Pax2-positive inhibitory interneuron precursors and MBP-positive oligodendroglial cells are located. In contrast, more stationary cells such as mature granule cells and Purkinje cells are associated with lower levels of hyaluronan in their environment. Thus, hyaluronan-rich fibers are concentrated at sites where specific neural precursor cell types migrate, and the anisotropic orientation of these fibers suggests that they may support guided neural migration during brain development.     

Progressive loss of neuronal src protein in postnatal weaver and staggerer cerebellum

Two forms of the c-src protein-tyrosine kinase, pp60c-src, are detectable in the central nervous system. One form pp60+, appears to be exclusively expressed in neurons and is characterized by insertion of 6 amino acids compared to its non-neuronal counterpart, pp60. These 2 proteins were studied in the mutant mouse strains weaver and staggerer with postnatal loss of cerebellar granular neurons. We found a continuous postnatal decline of the neuronal form of pp60c-src, pp60+, in the cerebellum of both mutants concomitant with the degeneration of cerebellar granule cells. This indicates that granular neurons provide the main source for pp60+ in the cerebellar cortex.     

Spatial and temporal changes in natural and target deprivation-induced cell death in the mouse inferior olive

The survival of inferior olive neurons is dependent on contact with cerebellar Purkinje cells. There is evidence that this dependence changes with time. Because inferior olivary axons, called climbing fibers, already show significant topographical ordering in cerebellar target zones during late embryogenesis in mice, the question arises as to whether olive neurons are dependent on target Purkinje cells for their survival at this early age. To better characterize this issue, inferior olive development was studied in two transgenic mouse mutants, wnt-1 and L7ADT, with embryonic and early postnatal loss of cerebellar target cells, respectively, and compared to that in the well-studied mutant, Lurcher. Morphological criteria as well as quantitative measures of apoptosis were considered in this developmental analysis. Survival of inferior olive neurons is observed to be independent of Purkinje cells throughout embryogenesis, but dependence begins immediately at birth in both wild types and mutants. Thereafter, wild types and mutants show a rapid increase in olive cell apoptosis, with a peak at postnatal day 4, followed by a period of low-level, but significant, apoptosis that continues to at least postnatal day 11; the main difference is that apoptosis is quantitatively enhanced in the mutants compared to wild types. The multiphasic course of these effects roughly parallels the known phases of climbing fiber synaptogenesis. In addition, despite significant temporal differences among the mutants with respect to absolute numbers of dying cells, there are common spatial features suggestive of distinct intrinsic programs linking different olivary subnuclei to their targets.