Cytotoxic T-lymphocyte tolerance to minor H-43a alloantigen is induced exclusively in the context of the self major histocompatibility complex class I H-2Kb molecules

We elucidated previously that cytotoxic T lymphocyte precursors (CTLp) against H-43a allo-antigen, which we had discovered as a new mouse minor H antigen, were primed in H-43b mice only in the context of self H-2Kb restriction element, and that anti-H-43a CTLp tolerance was induced in H-43b mice by injection with H-43a spleen cells (SC) from H-43 congenic mice, i.e., under the condition of disparity at only the H-43 locus. The present study attempted to determine whether the H-2Kb restriction element for anti-H-43a CTLp priming is also implicated in the induction of anti-H-43a CTLp tolerance. For this purpose, we used a newly established H-43b C3W (H-2k) strain which is H-43 congenic to H-43a C3H/HeN. When (C3W X B10.MBR)F1 (H-43b, H-2Kk/b, Ik/k, Dk/q) mice were injected with H-43a-bearing (C3H/HeN X B10.AKM)F1 (H-43a/b;H-2Kk/k,Ik/k,Dk/q)SC, their selfH-2Kb-restricted anti-H-43a CTLp were were primed (cross-priming). By contrast, injection of H-43a-bearing (C3H/HeN X B10.MBR)F1 (H-43a/b; H-2Kk/b,Ik/k, Dk/q)SC, which differ from (C3H/HeN x B10.AKM) F1 SC solely at H-2K and possess H-2Kb molecules, did not prime but specifically inactivated the anti-H-43a CTLp of (C3W x B10.MBR)F1 mice. These results indicate clearly that anti-H-43a CTLp tolerance is induced exclusively in the context of the H-2Kb element expressed on the antigenic H-43a SC.     

Molecular shape and ATP binding activity of rat p50, a putative mammalian homologue of RuvB DNA helicase

Based on partial amino acid sequences of p50 purified from a high-salt buffer extract of a rat liver nuclear matrix fraction, p50 cDNA was cloned and sequenced, and its amino acid sequence was predicted. The sequence contained helicase motifs, and showed homology with RuvB DNA helicase of Thermus thermophilus and an open reading frame for an unknown 50.5 k protein of Saccharomyces cerevisiae. p50 was expressed as a GST-fusion protein and antiserum against the protein was generated. p50 was localized to the nuclear matrix by cell fractionation and immunoblotting. p50 bound to ATP-Sepharose beads. Ultracentrifugation and gel filtration analyses showed that p50 in rat liver and Xenopus egg mitotic extracts exists as large complexes corresponding to 697 k and 447 k, respectively. A 50 k protein reactive with p50 antibodies was detected not only in rat liver nuclei, but also in a Xenopus egg cytoplasm fraction and a S. cerevisiae extract. This suggests that this putative DNA helicase is present in a wide variety of species ranging from yeast to mammals.     

Characteristics of DNA sequences in the sites of permanent attachment to the nuclear matrix located in the vicinity of replication initiation site

The permanent DNA attachment sites to the nuclear matrix in the domain of chicken alpha-globin genes originally found in erythrocyte nuclei are shown to exist in sperm and cultured fibroblast cells too. Short fragments of permanently attached to the nuclear matrix DNA have been cloned and sequenced. A primary structure of a 1.7 k.b. fragment from 5'-region of chicken alpha-globin gene domain containing both replication origin and permanent attachment site has been determined. A region possessing homologies with papovaviral replication origins and putative mammalian ARS elements has been found on the 1.7 k.b. fragment. A region containing short internal repeats and GC-rich motifs has also been found. Similar motifs were observed in several of the cloned short fragments of DNA permanently attached to the nuclear matrix.     

Cytotoxic T lymphocyte response to minor H-42a alloantigen in H-42b mice: clonal inactivation of the precursor cytotoxic T lymphocytes by veto-like spleen cells that express the H-42a antigen

When (B10.BR X CWB)F1 (BWF1; H-2k/b) mice carrying the H-42b allele at the minor H-42 locus were injected with H-42a C3H.SW (CSW; H-2b) or C3H (H-2k) spleen cells (SC), self-H-2Kb restricted anti-H-42a pCTL in the BWF1 recipients were primed and differentiated to anti-H-42a CTL after in vitro stimulation with (B10.BR X CSW)F1 (BSF1; H-2k/b, H-42b/a) SC. In contrast, anti-H-42a pCTL in H-42b mice were inactivated by injection with H-42-congenic H-42a SC, and stable anti-H-42a CTL tolerance was induced. Preference of H-2Kb restriction of anti-H-42a CTL was strict, and self-H-2Kb-restricted anti-H-42a CTL did not lyse target cells carrying H-42a antigen in the context of H-2Kbm1. Involvement of suppressor cells in the anti-H-42a CTL tolerance was ruled out by the present cell transfer study and the previous cell-mixing in vitro study. Notably, treatment with anti-Thy-1.2 antibody (Ab) plus complement (C) wiped out the ability of CSW SC in the priming of anti-H-42a pCTL of BWF1 mice but left that of C3H SC unaffected, and injection of the anti-Thy-1.2 Ab plus C-treated CSW SC induced anti-H-42a CTL tolerance in the BWF1 recipients. Furthermore, H-42a/b, I-Ab/bm12 [CSW X B6.CH-2bm12 (bm12)]F1 SC could not prime anti-H-42a pCTL in H-42b, I-Ab (CWB X B6)F1 recipients, whereas H-42a/b, I-Ab (CSW X B6)F1 SC primed anti-H-42a pCTL in H-42b, I-Ab/bm12 (CWB X bm12)F1 recipients. The unresponsiveness of anti-H-42a pCTL in H-42b mice to H-42-congenic H-42a SC was sometimes corrected by immunization of H-42b female mice with H-42-congenic H-42a male SC. Taking all of the results together, we propose the following. Unresponsiveness of anti-H-42a pCTL in H-42b mice to H-42-congenic H-42a SC is caused by "veto cells" contained in the antigenic H-42a SC. Anti-H-42a pCTL in the H-42b recipients directly interacting with H-42-congenic H-42a SC, which bear H-42a antigen and H-2Kb restriction element, are inactivated or vetoed.(ABSTRACT TRUNCATED AT 400 WORDS)     

DNA replication and the nuclear envelope

Upon isolation of the nuclear membrane from cultured mouse leukemia L5178Y cells, approximately 1% of the total nuclear DNA was found to be attached to this structure. After pulse labeling of DNA and isolation of the nuclear membrane, the ratio of labeled DNA in the membrane fraction and in the rest of chromatin was compared. Results indicate that with a 3 min pulse, DNA in the membrane fraction showed slightly higher specific activity, but when the pulse was longer than 5 min there was no difference in the specific activities. Since the DNA fragment associated with the membrane fraction was found to be long enough to contain most of the DNA labeled during a 5 min pulse, the results obtained indicate that there is no preferential association of DNA to the nuclear envelope during initiation or elongation of DNA.     

Active DNA transcription sites released from the genome of normal embryonic chicken cells.

Single-stranded DNA (ssDNA) isolated from (and amounting to 1.5-2% of) native nuclear DNA of cultured embryonic chicken cells labelled 1-2 days with 3H-thymidine was analyzed by self-hybridization, hydroxyapatite chromatography (HAC) partial digestion with S1 nuclease, isopycnic centrifugation. Two main fractions were rehybridized to excess amounts of bulk nuclear DNA or total cytoplasmic RNAs. The major fraction, equivalent to 75% of total ssDNA, consists of unique DNA sequences, apparently derived from multiple coding regions of the cell genome, since they are not self-reassociating but are hybridizable to the non repetitious portion of bulk nuclear DNA and 40-45% of them are complementary to cell RNAs. About half of these ssDNA sequences hybridizable to cell RNAs seem to be closely connected with molecules belonging to the minor ssDNA fraction. The latter fraction consists of self-reassociating, moderately repeated DNA sequences, mainly derived from non coding regions of the cell genome. These findings are discussed in the light of others, showing interspersion of coding and non coding DNA sequences and susceptibility of active genes to certain nucleasic attacks.     

Characterization of cytoplasmic DNA of mouse-myeloma cells.

Cytoplasmic DNA from mouse myeloma cells comprised between 1% and 2% of the total cellular DNA. Detergent-prepared cytoplasmic lysate consisted mainly of 8-S and 22-S species. While these DNA species were present in the 13000 times g pellet of the detergent-prepared cytoplasmic lysate, only the light DNA species was present in the 13000 times g supernatant fraction. In neutral CsCl gradients the DNA of both cytoplasmic fractions had a buoyant density of 1700 g/cm3, which is identical to that of nuclear DNA. The similarity between the cytoplasmic and nuclear DNA was also demonstrated by analysis on alkaline CsCl gradients. A small proportion of closed-circular DNA, presumably of mitochondrial origin, was demonstrated only in cytoplasmic fraction obtained from mechanically disrupted cells and not in detergent-prepared cytoplasmic lysate. It was found that poly (A)-containing mRNA and 28-S ribosomal RNA hybridized to about the same extent to the cytoplasmic DNA as compared to nuclear DNA. The results indicate that most of the cytoplasmic DNA in myeloma cells is similar to nuclear DNA and does not consist of mitochondrial DNA.     

Isolation and Characterization of Supercoiled Circular Deoxyribonucleic Acid from Beta-Hemolytic Strains of Escherichia coli

Covalently closed circular deoxyribonucleic acid (DNA) molecules were isolated by cesium chloride centrifugation in the presence of ethidium bromide from a naturally occurring beta-hemolytic Escherichia coli strain (SC52). The open circular forms have contour lengths of 2.25 ± 0.1 μm, 24.0 ± 0.3 μm, and 29.5 ± 0.5 μm. The beta-hemolytic character of E. coli SC52 can be transferred by conjugation to a nonhemolytic recipient strain. Analysis of the supercoiled DNA of the hemolytic recipient demonstrated that the two large supercoiled DNA molecules of E. coli SC52 are transferred during this event, too. A beta-hemolytic laboratory E. coli strain and several of its derivatives have been shown to contain at least one circular DNA molecule, slightly larger in size than those isolated from E. coli SC52 and its conjugant. The possible significance of these DNA molecules for hemolysin production and transfer is discussed.     

Physico-chemical determination of the ploidy of the unicellular alga, Chlorella pyrenoidosa (strain 211/8b) (author's transl)]

The ploidy of the unicellular green alga Chlorella pyrenoidosa (strain 211/8b) has been determined by means of renaturation kinetics. The nuclear DNA is made up from fast, intermediate and slow renaturing sequences, which represent respectively about 5, 15 and 80% of the DNA. These observations are consistent with the findings in other eukaryotic nuclear DNAs. Nevertheless, the relative importance of the repeated sequences is much lower than that observed in Chlamydomonas reinhardi [16] and in higher plants [18-20], but slightly higher than that obtained in Chlorella vulgaris [17]. The kinetic complexity of the main fraction of the Cl. pyrenoidosa nuclear DNA is found to be 2.94 - 10-10 daltons (mean value of five independant experiments) assuming value of 2.1 - 10-8 daltons for Cl. pyrenoidosa chloroplastic DNA. When compared with the analytical complexity of this fraction (80% of the nuclear DNA analytical complexity, that is 2.02 - 10-10 daltons), one can assume that the slow renaturing fraction of the nuclear DNA is constituted by a unique nucleotide sequence. This result thus suggests that Cl. pyrenoidosa (strain 211/8b) is an haploid organism. The possible existence of an haploid genome in the nuclei of the algae from Chlorella genus and the apparent absence of sexuality might explain the high discrepancy observed in the G + C content of the Chlorella nuclear DNAs.     

Escherichia coli plasmid pBR313 insertion into plant protoplasts and into their nuclei

Summary Cowpea mesophyll protoplasts were shown to bind irreversibly up to 3% input radioactive pBR313 plasmid DNA after 15 min of contact. Maximum uptake occurred in the presence of 5mM ZnSO₄ and 5 g/ml poly-L-ornithine. Under these conditions about one half of the TCA precipitable radioactivity was associated with the nuclear fraction and behaved as linear plasmid molecules. These could not be chased from the protoplasts upon further incubation with unlabeled plasmid DNA. The presence of donor DNA within the nuclear fraction is most probably not due to an artifactual redistribution of adsorbed plasmid DNA. Prolonged incubation periods resulted in extensive degradation of plasmid in the incubation medium but little degradation occurred in the protoplasts. The donor DNA was not covalently associated with the protoplast nuclear DNA.     

Diversity of sequences in total and polyadenylated nuclear RNA from Drosophila cells.

Complementary DNA was synthesized using polyadenylated nuclear RNA of cultured Drosophila cells as template. The kinetics of hybridization of this cDNA with nuclear RNA indicated that the complexity of this RNA population is five to ten times greater than that of cytoplasmic mRNA. The same difference in the fraction of DNA represented was obtained when nuclear and cytoplasmic RNA were hybridized with labeled unique sequence DNA. The fraction of the DNA sequences represented in total number of polyadenylated nuclear RNA is much higher than that represented in cytoplasmic RNA.     

Nuclear membrane lipid peroxidation products bind to nuclear macromolecules

Ascorbate-Fe2+-driven lipid peroxidation processes in isolated rat liver nuclei give rise to products that bind to DNA and total nuclear proteins. This has been demonstrated by integrating [3H]arachidonic acid into the nuclear membranes. Lipid peroxidation was estimated from the formation of 2-thiobarbituric acid chromophore, and from the relative distribution of 3H-peroxidation products between the lipidic fraction and the nonlipidic fraction of the nuclear suspensions during incubation. The amount of 3H-peroxidation products associated with DNA and total nuclear proteins increased about threefold, when compared to control experiments (no ascorbate-Fe2+), after 180 min of incubation. In contrast, the radioactivity associated with the histone fraction was observed to decrease during incubation. The positive correlation obtained between the formation of thiobarbituric acid chromophore and the association of radioactivity with DNA and nuclear proteins indicates that the binding processes were dependent on peroxidation of the nuclear membrane lipids.     

Rapid Isolation of Rat Brain Nuclei on Percoll Gradients

A rapid isotonic method for fractionation of nuclei from rat brain is described. This procedure is based on the use of discontinuous colloidal silica gel (Percoll) gradients. We start from a 63,000-g purified nuclear pellet (fraction P3) isolated from gray matter and white matter separately. This is followed by fractionation of fraction P3 in an initial differential centrifugation step on five-step Percoll gradients producing six nuclear fractions designated 1, 2, 3 (gray matter) and 4, 5, 6 (white matter). Fractions 2, 4, and 5 obtained from this centrifugation are heterogeneous. These fractions are subfractionated further under isopycnic conditions using five-step Percoll gradients to yield subfractions 2b, 4b, and 5c. Three methods were used to characterize the nuclear types. First, light and electron microscopic examination was used to identify the nuclei in each preparation and to assess the purity of each preparation. Second, the activities of RNA polymerase I and II were monitored. Third, the protein/DNA ratios of the nuclear fractions were determined. Fraction 1 was enriched in neuronal nuclei; fractions 2b and 4b in astrocytic nuclei; and fractions 3, 5c, and 6 in nuclei of oligodendrocytes. RNA polymerase I and II activity was highest in fraction 1, which also displayed the highest protein/DNA ratio. Electron microscopy showed that the various classes of nuclei are congruent to 90% pure. Therefore, the procedure described here is suitable for obtaining highly purified neuronal and three types of glial nuclei from rat brain.     

Effect of nickel(II) on DNA-protein binding,thymidine incorporation,and sedimentation pattern of chromatin fractions from intact mammalian cells

Nuclear uptake and chromatin binding of nickel(II) was investigated in Chinese hamster ovary (CHO) cells. The cytoplasmic:nuclear ratio of nickel immediately following treatment was 5:1, but by 24 and 48 hours this ratio decreased to 4:l and 2:1, respectively, indicating that nickel is retained longer in the nucleus than cytoplasmic nickel. Chromatin was fractionated by sonication and centrifugation into fast-sedimenting, magnesium-insoluble, or magnesiumsoluble components. The magnesium-insoluble portion bound more nickel ions and retained the metal longer than either the magnesium-soluble or the fastsedimenting fractions. Treatment of cells with nickel chloride (NiCl₂) decreased the amount of DNA in the magnesium-insoluble fraction but increased the amount of DNA in the fast- sedimenting chromatin fraction. The magnesium-insoluble fraction isolated from nickel-treated cells contained approximately ten times more [35-S]-methionine–labeled protein per milligram DNA compared with untreated cells. The magnesium-soluble and the fast-sedimenting fractions isolated from the nickel-treated cells did not exhibit a similar increase in [35-S]-methionine–labeled protein per milligram of DNA. Nickel treatment suppressed [14-C]-thymidine incorporation into total DNA by 30% compared with untreated cells. However, the magnesium-insoluble chromatin fraction from nickel-treated cells had a tenfold to 20-fold increase in thymidine incorporation, while the other chromatin fractions did not exhibit an increase in thymidine incorporation. These findings indicate that nickel induced widespread alterations in chromatin conformation and preferentially interacted with an Mg-insoluble component of chromatin.     

A novel class of nuclear RNA (nRNA) with a long life span as a gene repressor candidate.

Different systemic organs of fetal mice were continuously labelled with 5-3H-uridine during the organogenesis periods, and chased for various lengths of time after birth. In the autoradiographs made on paraffin-embedded sections of the organs taken after the chase for longer periods than 1 week, including a 12-months chase, specific labels were present exclusively in all the nuclei. The specific nuclear labels were resistant to RNase A or H digestion and to acid hydrolysis with 1 N HCl at 60 degrees C for 5 min, but were completely abolished by DNase digestion or prolonged acid hydrolysis for 10 min, the optimum condition for the Feulgen reaction to stain DNA. Electrophoretic analysis of the total nucleic acids extracted from the different organs chased for 3 or 12 months showed all the tritium radioactivity to be present in the DNA fraction before digestion with DNase or RNase A, and to be completely absent from the DNA fraction and shifted to the RNA fraction, or to be largely destroyed by degradation, after each digestion, respectively. By HPLC analysis of the total nucleic acid extract after further successive digestions with nuclease P1 and alkaline phosphatase into the constituent nucleotides, it was shown that all tritium activity was incorporated in uridine, without any detectable label in other nucleotides. By the simultaneous labeling of human peripheral lymphocytes at the late S-phase with 5-3H-uridine and BrdU, it was demonstrated that the autoradiographic labels, which, this time, were labile to RNase A digestion, were present in the G-bands of the spread chromosomes as identified by BrdU immunohistochemistry. The findings strongly indicate the presence of a novel class of nuclear RNA (nRNA). This type of RNA (a) may be localized in the nucleus in close association or hybridization with nuclear DNA, (b) have a life span as long as that of the cell, and (c) be duplicated in the late phase of DNA replication. The nRNA may play a fundamental role as gene repressor existing in the G-bands of metaphase chromosomes in the process of ontogeny and cytodifferentiation.     

Non-random distribution of the Ty1 elements within nuclear DNA of Saccharomyces cerevisiae

Ty1 homologous sequences appear to be non-randomly distributed among different density classes of nuclear yeast DNA. Characteristic patterns of Ty1 containing EcoRI fragments can be generated from the various DNA fractions. The sequences are particularly enriched in the A + T rich part of the main nuclear DNA fraction, while the frequency in the rDNA containing heavy satellite DNA is low. The transposon however, seems to be present in this dense fraction, at least for some strains.     

Localization of DNA polymerase α on the nuclear membrane in sea urchin embryos

Subnuclear localization of DNA polymerase α was studied in sea urchin embryos. Blastula nuclei treated with EDTA and potassium phosphate released subnuclear components bearing most of the nuclear DNA polymerase α. These components were suggested to be a part of nuclear membrane based on their buoyant densities (1.177 and 1.136 g/cm³) in isopyknic centrifugation and the nuclear pore-like structure. Contamination with DNA and endoplasmic reticulum membrane to the subnuclear components was shown to be negligible. These results suggested that DNA polymerase α associates with nuclear membrane of sea urchin embryos. Nuclear membrane deprived of DNA polymerase α was able to associate with nuclear DNA polymerase α from blastulae and the cytoplasmic enzyme of unfertilized eggs efficiently, but not with the cytoplasmic enzyme of gastrulae. This result suggests that the nuclear membrane is originates from the endoplasmic reticulum with which DNA polymerase α associates in unfertilized eggs.     

Changes in the composition of DNA-bound nonhistone protein in the thymus and liver of gamma-irradiated rats

A supramolecular DNA complex (SC DNA) and DNA of a phenol nuclear matrix (PNM DNA) were extracted, by the phenol method, from rat thymus and liver 15 min following 10 Gy gamma-irradiation. The method of electrophoresis in polyacrylamide gel was used to study a composition of nonhistone proteins firmly bound to these DNA fractions. Irradiation was shown to induce the occurrence of new proteins and redistribution of proteins between SC DNA and PNN DNA of rat organs.