Selective affinity fractionation of murine cytotoxic T lymphocytes (CTL). Unique lectin specific binding of the CTL associated surface glycoprotein, T 145.

We have analyzed the lectin binding characteristics of cytotoxic T lymphocyte (CTL)-derived surface labeled glycoproteins by affinity chromatography of the labeled glycoproteins on a panel of immobilized lectin adsorbents. Evidence is presented for the specific interaction of the CTL-associated glycoprotein T 145 with a lectin derived from Vicia villosa seeds. Conditions are described for the preparation and use of lectin affinity adsorbents for the rapid isolation of T 145 bearing cytotoxic T lymphocytes. Direct proof is given to show that T 145-positive cells arising from a variety of T-cell activations constitute the only subpopulation of cells with ability to perform cell-mediated T-cell cytotoxicity. Specific depletion of the CTLs by adherence to V. villosa adsorbents is shown by their depletion in the nonbound cell fraction and correspondingly enriched recovery in the sugar eluted cell fraction. Specific affinity fractionation of CTLs has occurred in every strain combination tested and irrespective of the actual antigen specificity of the effector cell population.

Histones and G banding of chromosomes.

Polylysine, polyarginine, and histones H1, H2A, H2B, and H3 inhibit Giemsa staining and chromosome banding by binding to DNA and preventing side stacking of the positively charged thiazine dyes to the negatively charged phosphate groups on DNA. This is a nonspecific effect and does not of itself provide evidence for a role of histones in G banding. The question of whether histones are involved in chromosome banding is reviewed.

DNA repair rates mapped along the human PGK1 gene at nucleotide resolution.

The repair of cyclobutane pyrimidine dimers (CPDs), DNA lesions induced by ultraviolet light, was studied at nucleotide resolution. Human fibroblasts were irradiated with ultraviolet light and allowed to repair. The DNA was enzymatically cleaved at the CPDs, and the induced breaks along the promoter and exon 1 of the PGK1 gene were mapped by ligation-mediated polymerase chain reaction. Repair rates within the nontranscribed strand varied as much as 15-fold, depending on nucleotide position. Preferential repair of the transcribed strand began just downstream of the transcription start site but was most pronounced beginning at nucleotide +140 in exon 1. The promoter contained two slowly repaired regions that coincided with two transcription factor binding sites.

Replication timing of genes and middle repetitive sequences.

DNA replication in mammals is temporally bimodal. "Housekeeping" genes, which are active in all cells, replicate during the first half of the S phase of cell growth. Tissue-specific genes replicate early in those cells in which they are potentially expressed, and they usually replicate late in tissues in which they are not expressed. Replication during the first half of the S phase is, therefore, a necessary but not sufficient condition for gene transcription. A change in the replication timing of a tissue-specific gene appears to reflect the commitment of that gene to transcriptional competence or to quiescence during ontogeny. Most families of middle repetitive sequences replicate either early or late. These data are consistent with a model in which two functionally distinct genomes coexist in the nucleus.

Binding of transcription factors creates hot spots for UV photoproducts in vivo.

Cyclobutane dipyrimidines and less than mean value of 6-4 dipyrimidines are the two major classes of mutagenic DNA photoproducts produced by UV irradiation of cells. We developed a method to map cyclobutane dipyrimidines at the DNA sequence level in mammalian cells. The frequency of this class of photoproducts was determined at every dipyrimidine along the human phosphoglycerate kinase-1 (PGK1) promoter sequence and was compared to the UV-induced frequency distribution of mean value of 6-4 dipyrimidines. After irradiation of living cells containing active or inactive PGK1 genes, enzymatic or chemical cleavage at UV photoproducts, and amplification by ligation-mediated polymerase chain reaction, photofootprints were seen in all regions which bind transcription factors and appear as DNase I footprints. Photoproduct frequency within transcription factor binding sites was suppressed or enhanced relative to inactive genes or naked DNA with enhancements of up to 30-fold. Since photoproducts are mutagenic, this indicates that photoproduct (mutation?) hot spots may be tissue specific in mammals.

Chromosome organization and chromatin modification: influence on genome function and evolution.

Histone modifications of nucleosomes distinguish euchromatic from heterochromatic chromatin states, distinguish gene regulation in eukaryotes from that of prokaryotes, and appear to allow eukaryotes to focus recombination events on regions of highest gene concentrations. Four additional epigenetic mechanisms that regulate commitment of cell lineages to their differentiated states are involved in the inheritance of differentiated states, e.g., DNA methylation, RNA interference, gene repositioning between interphase compartments, and gene replication time. The number of additional mechanisms used increases with the taxon's somatic complexity. The ability of siRNA transcribed from one locus to target, in trans, RNAi-associated nucleation of heterochromatin in distal, but complementary, loci seems central to orchestration of chromatin states along chromosomes. Most genes are inactive when heterochromatic. However, genes within beta-heterochromatin actually require the heterochromatic state for their activity, a property that uniquely positions such genes as sources of siRNA to target heterochromatinization of both the source locus and distal loci. Vertebrate chromosomes are organized into permanent structures that, during S-phase, regulate simultaneous firing of replicon clusters. The late replicating clusters, seen as G-bands during metaphase and as meiotic chromomeres during meiosis, epitomize an ontological utilization of all five self-reinforcing epigenetic mechanisms to regulate the reversible chromatin state called facultative (conditional) heterochromatin. Alternating euchromatin/heterochromatin domains separated by band boundaries, and interphase repositioning of G-band genes during ontological commitment can impose constraints on both meiotic interactions and mammalian karyotype evolution.

Mutational spectrum analysis of RNase H(35) deficient Saccharomyces cerevisiae using fluorescence-based directed termination PCR.

Mutational spectrum analysis has become an informative genetic tool to understand those protein functions involved in mutation avoidance pathways since specific types of mutations are often associated with particular protein defects involved in DNA replication and repair. In this study, we describe a novel, fluorescence-based procedure for direct determination of deletions and insertions with 100% accuracy. We performed two complementary directed termination PCR with near infrared dye-labeled primers, followed by visualization of termination fragments using an automated Li-cor DNA sequencer. This method is used for rapid analysis of mutational spectra generated in nuclease-defective strains of Saccharomyces cerevisiae to elucidate the role of RNase H(35) in RNA primer removal during DNA replication and in mutation avoidance. Strains deficient in RNH35 displayed a distinct spontaneous mutation spectrum of deletions characterized by a unique 4 bp deletion in a lys2-Bgl allele. This was in sharp contrast to strains deficient in rad27 that displayed duplication mutations. Further analysis of mutations in a rnh35/rad27 double mutant revealed a mixed spectrum. These results indicate that RNase H(35) may participate in a redundant pathway in Okazaki fragment processing and that mutational spectra caused by protein deficiencies may be more intermediate-specific than pathway-specific.

Fluorescence-based directed termination PCR: direct mutation characterization without sequencing.

We describe a fluorescence-based directed termination PCR (fluorescent DT-PCR) that allows accurate determination of actual sequence changes without dideoxy DNA sequencing. This is achieved using near infrared dye-labeled primers and performing two PCR reactions under low and unbalanced dNTP concentrations. Visualization of resulting termination fragments is accomplished with a dual dye Li-cor DNA sequencer. As each DT-PCR reaction generates two sets of terminating fragments, a pair of complementary reactions with limiting dATP and dCTP collectively provide information on the entire sequence of a target DNA, allowing an accurate determination of any base change. Blind analysis of 78 mutants of the supF reporter gene using fluorescent DT-PCR not only correctly determined the nature and position of all types of substitution mutations in the supF gene, but also allowed rapid scanning of the signature sequences among identical mutations. The method provides simplicity in the generation of terminating fragments and 100% accuracy in mutation characterization. Fluorescent DT-PCR was successfully used to generate a UV-induced spectrum of mutations in the supF gene following replication on a single plate of human DNA repair-deficient cells. We anticipate that the automated DT-PCR method will serve as a cost-effective alternative to dideoxy sequencing in studies involving large-scale analysis for nucleotide sequence changes.

Metal ion-dependent hydrogen peroxide-induced DNA damage is more sequence specific than metal specific.

The frequency of oxidative base damage along the human p53 and PGK1 genes was determined at nucleotide resolution by cleaving DNA at oxidized bases with endonuclease III and formamidopyrimidine DNA glycosylase and then using the ligation-mediated PCR technique to map induced break frequency. Damage was induced either in vivo by exposing cultured human male fibroblasts to H2O2 or in vitro by exposing purified genomic DNA to H2O2 plus ascorbate in the presence of Cu(II), Fe(III), or Cr(VI) metal ions. All four base damage patterns from either in vivo or in vitro treatments were nearly identical in both regions of the genome. The frequency of base damage varied along the DNA, with guanine being the most commonly damaged base. In the Fe(III)-mediated in vitro reactions, single-stranded breaks were almost completely suppressed by addition of sucrose, which facilitated mapping of base damage. The in vitro base damage pattern generated by Cr(VI), ascorbate, and H2O2 was similar to that of the other metal ions, with the exception of several unique positions; these were heavily damaged only in the presence of Cr(VI). Isolated nuclei suffered little oxidative base damage in the presence of ascorbate and H2O2, and we conclude that during H2O2 in vivo treatment of cells, metal ions (or metal-like ligands) are freed from the cytoplasm to migrate into the nucleus and supply the redox cycling ligands necessary for oxidative base damage. These data simplify the complexity of H2O2-induced oxidative damage and mutagenesis studies by demonstrating the commonality of damage catalyzed by different transition metal ions and by showing that the pattern of H2O2-mediated oxidative base damage is determined almost entirely by the primary DNA sequence, with chromatin structure having a limited effect. Our data suggest a model for base damage in which DNA-metal ion binding domains can equally accommodate a variety of different metal ions and thus are a key factor in determining the local probability of DNA damage.

Replication time of interspersed repetitive DNA sequences in hamsters.

The replication time of 34 hamster genomic DNA segments containing interspersed repeat sequences was determined by probing the cloned segments with nick-translated early- and late-replicating hamster DNA. One-third of these cloned families replicated early, one-third replicated late, and one-third replicated without temporal bias. 19 different inserts from these clones along with the SINE, Alu, and the LINE, A36Fc, were used to probe Southern blots of early- and late-replicating hamster or human DNA. We report long interspersed repeats, LINEs, are selectively partitioned into late-replicating DNA and are often concertedly hypomethylated, while short interspersed repeats, SINEs, are selectively partitioned into early-replicating DNA. For some interspersed repeat families, this partitioning is complete or almost complete. The CCGG frequency is very low in late-replicating DNA. The mammalian chromosome's pattern of early-replicating R-bands and late-replicating G-bands reflects a differential distribution of LINEs and SINEs.

Heterogeneous repair of N-methylpurines at the nucleotide level in normal human cells.

Base excision repair rates of dimethyl sulfate-induced 3-methyladenine and 7-methylguanine adducts were measured at nucleotide resolution along the PGK1 gene in normal human fibroblasts. Rates of 7-methylguanine repair showed a 30-fold dependence on nucleotide position, while position-dependent repair rates of 3-methyladenine varied only sixfold. Slow excision rates for 7-methylguanine bases afforded the opportunity to study their excision in vitro as a model for base excision repair. A two-component in vitro excision system, composed of human N-methylpurine-DNA glycosylase (MPG protein) and dimethyl sulfate-damaged DNA manifested sequence context-dependent rate differences for 7-methylguanine of up to 185-fold from position to position. This in vitro system reproduced both the global repair rate, and for the PGK1 coding region, the position-dependent repair patterns observed in cells. The equivalence of in vivo repair and in vitro excision data indicates that removal of 7-methylguanine by the MPG protein is the rate-limiting step in base excision repair of this lesion. DNA "repair rate footprints" associated with DNA glycosylase accessibility were observed only in a region with bound transcription factors. The "repair rate footprints" represent a rare chromatin component of 7-meG base excision repair otherwise dominated by sequence-context dependence. Comparison of in vivo repair rates to in vitro rates for 3-methyladenine, however, shows that the rate-limiting step determining position-dependent repair for this adduct is at one of the post-DNA glycosylase stages. In conclusion, this study demonstrates that a comparison of sequence context-dependent in vitro reaction rates to in vivo position-dependent repair rates permits the identification of steps responsible for position-dependent repair. Such analysis is now feasible for the different steps and adducts repaired via the base excision repair pathway.

Cupric ion/ascorbate/hydrogen peroxide-induced DNA damage: DNA-bound copper ion primarily induces base modifications.

The kinetics of frank DNA strand breaks and DNA base modifications produced by Cu(II)/ascorbate/H2O2 were simultaneously determined in purified human genomic DNA in vitro. Modified bases were determined by cleavage with Escherichia coli enzymes Nth protein (modified pyrimidines) and Fpg protein (modified purines). Single-stranded lesion frequency before (frank strand breaks) and after (modified bases) Nth or Fpg protein digestion was quantified by neutral glyoxal gel electrophoresis. Dialysis of EDTA-treated genomic DNA purified by standard proteinase K digestion/phenol extraction was necessary to remove low molecular weight species, probably transition metal ions and metal ion chelators, which supported frank strand breaks in the presence of ascorbate + H2O2 without supplemental copper ions. We then established a kinetic model of the DNA-damaging reactions caused by Cu(II) + ascorbate + H2O2. The principal new assumption in our model was that DNA base modifications were caused exclusively by DNA-bound Cu(I) and frank strand breaks by non-DNA-bound Cu(I). The model was simulated by computer using published rate constants. The computer simulation quantitatively predicted: (1) the rate of H2O2 degradation, which was measured using an H2O2-sensitive electrode, (2) the linearity of accumulation of DNA strand breaks and modified bases over the reaction period, (3) the rate of modified base accumulation, and (4) the dependence of modified base and frank strand production on initial Cu(II) concentration. The simulation significantly overestimated the rate of frank strand break accumulation, suggesting either that the ultimate oxidizing species that attacks the sugar-phosphate backbone is a less-reactive species than the hydroxyl radical used in the model and/or an unidentified hydroxyl radical-scavenging species was present in the reactions. Our experimental data are consistent with a model of copper ion-DNA interaction in which DNA-bound Cu(I) primarily mediates DNA base modifications and nonbound Cu(I) primarily mediates frank strand break production.

An enzyme-linked immunosorbent assay for measuring anti-sheep erythrocyte antibodies.

A simple ELISA assay for detecting murine anti-SRBC antibodies of IgG class was developed and the variation of the results according to different experimental conditions was investigated. Erythrocytes were left to settle in flexible plastic microtiter plates, after which they were fixed with glutaraldehyde and the remaining binding sites in the plates saturated with ovalbumin. Serum or monoclonal IgG antibodies were then allowed to react with the erythrocytes. Protein A coupled to alkaline phosphatase caused a color change in the subsequently added enzyme substrate. The results proved to be of good reproducibility, specificity and sensitivity. The assay can be used for measuring IgG concentration, estimating antibody avidity and number of antigenic determinants on the SRBC, as well as screening IgG anti-SRBC hybridomas. The precision of concentration estimates was very good when standard curves were used.

Identification of a Plasmodium chabaudi antigen present in the membrane of ring stage infected erythrocytes.

A novel antigen of asexual blood stages of the rodent malaria parasite Plasmodium chabaudi, was detected by means of a modified indirect immunofluorescence assay (IFA), using glutaraldehyde fixed and air dried monolayers of P. chabaudi infected erythrocytes. P. chabaudi hyperimmune sera gave a distinct surface immunofluorescence of erythrocytes infected with early stages of the parasite. Fixation and drying of the erythrocytes was necessary for the antigenic activity to be exposed. The antigens were species specific as P. chabaudi hyperimmune serum only stained P. chabaudi but not P. yoelii or P. falciparum infected erythrocytes. The antigenic activity involved in the IFA was resistant to trypsin, phospholipases and neuraminidase but not to pronase, suggesting that the antigens were polypeptides. The surface immunofluorescence was inhibited by an extract of parasitized erythrocytes, but not by similar extracts of normal erythrocytes. The inhibitory antigens were soluble and heat stable (100 degrees C, 5 min). For identification and characterization of the antigens, antibodies were isolated by acid elution from monolayers of infected erythrocytes and monoclonal antibodies were produced. Probing in immunoblotting of extracts of parasitized erythrocytes separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis with the eluted antibodies, showed that they reacted consistently with a polypeptide of Mr 105 000 (Pch105). The Pch105 antigen shares many characteristics with Pf155, a P. falciparum antigen considered as a candidate for a vaccine against that parasite.

Cloning of genomic fragment from Plasmodium chabaudi expressing a 105 kilodalton antigen epitope.

The 105 kDa antigen of Plasmodium chabaudi has many characteristics of the P. falciparum Pf 155 (RESA) molecule. A clone (pPC105e) from a P. chabaudi genomic library was isolated using immune screening with a 105 kDa antigen specific monoclonal antibody (B7E10). Southern, Northern and Western blotting analyses provide evidence for a lack of variability at the protein and DNA levels. A subclone of the insert in the expression vector pEX2, synthesises a fusion peptide which contains the epitope recognized by B7E10. Sequences homologous to the insert were detected in the genome of three other rodent and two primate malarias.

Repair rates of R-band, G-band and C-band DNA in murine and human cultured cells.

Repair of cyclobutane pyrimidine dimers (CPDs) in cultured neonatal human fibroblasts and in Mus spretus x M. castaneus F1 neonatal skin fibroblasts was analyzed after UVC-irradiation by cleavage with T4 endonuclease V cyclopyrimidine dimer glycosylase, alkaline-agarose gel electrophoresis, and Southern blotting. The blots were sequentially probed with 32P-labeled Alu, or B2, to preferentially illuminate R-band DNA, by L1 to preferentially illuminate G-band DNA, and by satellite DNA to illuminate C-band DNA. These three different DNA populations showed slightly different global nucleotide excision repair rates that are in the order of speed, R-band DNA > G-band DNA > C-band DNA. Fibroblasts from out-bred neonatal mice and humans showed similar band-specific repair rate ratios and the global repair rate of murine fibroblasts was almost as rapid as that of the human fibroblasts. The mass distribution of the human Alu-probed signal was further analyzed. Gel mobility data was fitted to a logistic equation to include all M(r) values. Hypothetical distributions of DNA randomly cleaved to a particular number-average molecular weight were fit to the logistic gel mobility function to determine how such a randomly cleaved distribution of a particular cleavage frequency would be displayed along the experimental gel. This revealed a rapidly repaired kinetic fraction that represented 17% of the Alu-probed signal (R-band DNA), almost none of the L1 probed signal (G-band DNA), and reflects transcription coupled repair of active genes. The remaining Alu-probed DNA showed a random distribution of UVC-induced CPDs throughout all stages of global nucleotide excision repair. The Alu-probed CPDs disappeared with an excellent fit to first order kinetics and with a half-life of seven hours.

Ligation-mediated PCR: robotic liquid handling for DNA damage and repair.

The investigation of in vivo DNA repair in mammalian cells at nucleotide resolution requires the quantification of break frequencies less than one per kilobase. By optimizing several parameters of the ligation-mediated PCR technique, we find that the required sensitivity can be achieved. We also report details of a one-day procedure that can be performed either with or without a robotic liquid handling workstation. The use of near-infrared fluorescent-labeled primers with detection by a LI-COR DNA sequencer provides for safe, nonradioactive detection, similar in sensitivity to the use of 32P-labeled primers but with the additional advantage that high-quality digitized data are obtained directly. Multiplexing can be performed; that is, more than one sequence can be analyzed in a single reaction, and multiple reactions can be processed robotically. Primer sets for exons 5-8 of the tumor suppressor gene, p53, were designed for simultaneous thermal cycling. The improved procedure with infrared detection was used to monitor low-frequency damage (<1 break/kb) and/or repair of UVB, UVC, and chemical methylation. Quantitative data on the linearity of response and reproducibility are described. The coefficient of variation for technical replicates was typically 10%. The methods described here will permit high sample throughput for the detection of DNA damage and repair as well as in vivo protein footprints.

Effect of hypophysectomy on alpha-melanotropin in discrete regions of the rat brain.

The effect of hypophysectomy on alpha-melanotropin (alpha MSH) concentrations in discrete brain regions was investigated. Hypophysectomy resulted in a 38-69% decrease in alpha MSH concentration in alpha MSH terminal regions 4 weeks after surgery. In contrast, the alpha MSH concentration in the arcuate nucleus, site of alpha MSH containing perikary, was unaffected by hypophysectomy. These results indicate that the brain alpha MSH system is distinct from, but related to that of the pituitary.

Repair of DNA alkylation damage.

Alkylation damage of DNA is one of the major types of insults which cells must repair to remain viable. One way alkylation damaged ring nitrogens are repaired is via the Base Excision Repair (BER) pathway. Examination of mutants in both BER and Nucleotide Excision Repair show that there is actually an overlap of repair by these two pathways for the removal of cytotoxic lesions in Escherichia coli. The enzymes removing damaged bases in the first step in the BER pathway are DNA glycosylases. The coding sequences for a number of methylpurine-DNA glycosylases (MPG proteins) were cloned, and a comparison of the amino-acid sequences shows that there are some similarities between these proteins, but nonetheless, compared to other DNA glycosylases, MPG proteins are more divergent. MPG proteins have been purified to homogeneity and used to identify their substrates ranging from methylating agents to deamination products to oxidatively damaged bases. The ligation-mediated polymerase chain reaction has been used to study the formation of alkylation damage, and its repair in mammalian cells. We have studied DNA damage in the PGK1 gene for a series of DNA alkylating agents including N-methyl-N'-nitro-N-nitrosoguanidine, Mechlorethamine, and Chlorambucil and shown that the damage observed in the PGK1 (phosphoglycerate kinase 1) gene depends on the alkylating agent used. This report reviews the literature on the MPG proteins, DNA glycosylases removing 3-methyladenine, and the use of these enzymes to detect DNA damage at the nucleotide level.