Processing of mispaired and unpaired bases in heteroduplex DNA in E. coli

Bacteriophage lambda and phi X 174 DNAs, carrying sequenced mutations, have been used to construct in vitro defined species of heteroduplex DNA. Such heteroduplex DNAs were introduced by transfection, as single copies, into E. coli host cells. The progeny of individual heteroduplex molecules from each infective center was analyzed. The effect of the presence of GATC sequences (phi X 174 system) and of their methylation (lambda system) was tested. The following conclusions can be drawn: some mismatched base pairs trigger the process of mismatch repair, causing a localized strand-to-strand information transfer in heteroduplex DNA: transition mismatches G:T and A:C are efficiently repaired, whereas the six transversion mismatches are not always readily recognized and/or repaired. The recognition of transversion mismatches appears to depend on the neighbouring nucleotide sequence; single unpaired bases (frameshift mutation "mismatches") are recognized and repaired, some equally efficiently on both strands (longer and shorter), some more efficiently on the shorter (-1) strand; large non-homologies (about 800 bases) are not repaired by the Mut H, L, S, U system, but some other process repairs the non-homology with a relatively low efficiency; full methylation of GATC sequences inhibits mismatch repair on the methylated strand: this is the chemical basis of strand discrimination (old/new) in mismatch correction; unmethylated GATC sequences appear to improve mismatch repair of a G:T mismatch in phi X 174 DNA, but there may be some residual mismatch repair in GATC-free phi X 174, at least for some mismatches.(ABSTRACT TRUNCATED AT 250 WORDS)     

Target organ and time-course in the mutagenicity of five carcinogens in MutaMouse: a summary report of the second collaborative study of the transgenic mouse mutation assay by JEMS/MMS.

We studied five carcinogens for (a) organ-specific mutagenicity and expression time in the transgenic (TG) mouse mutation assay and (b) clastogenicity in the peripheral blood micronucleus assay in the same mice. Groups of mice were injected intraperitoneally (ip) with N-nitroso-di-n-propylamine (NDPA), propylnitrosourea (PNU), 7, 12-dimethylbenz[a]anthracene (DMBA), 4-nitroquinoline-1-oxide (4NQO), or procarbazine (PCZ); 4NQO was also administered orally. LacZ mutant frequencies (MF) of various organs, sampled 7, 14 and 28 days after treatment, were analyzed by galE positive selection. At least 5 organs were analyzed in each experiment. Bone marrow, liver, and testis were always analyzed, as were each chemical's target organs. All chemicals, except NDPA, induced micronuclei. All chemicals increased lacZ MF in all of their target organs for carcinogenesis and, to a lesser extent, in some non-target organs. That suggests that an organ that has a positive response to a chemical in the TG mouse mutation assay is likely to develop tumors on exposure to that chemical, but it does not always happen. The time-course of MF increases (7-28 days) differed among tissues. In general, time-dependent increase in MF occurred in organs with a low cell proliferation rate whereas no increase, or even a decrease, occurred in organs with a high proliferation rate. Our results demonstrated that the TG mouse mutation assay is effective for the detection of chemical mutagenesis in the target organs for carcinogenesis, and organ and time-course variations in chemical mutagenesis are important issues for the establishment of an optimal protocol for the assay.     

The complexity of radiation-induced DNA damage as revealed by exposure to cell extracts.

The rejoining of single-strand breaks (SSBs) induced in plasmid DNA in the presence of 10 mmol dm(-3) Tris scavenger by aluminum K (Al(K)) ultrasoft X rays has been compared with that for SSBs induced by gamma radiation. The Al(K) ultrasoft X rays interact to produce low-energy secondary electrons, which are thought to be the main contributors to the formation of complex damage by low-LET radiations. The rejoining of radiation-induced SSBs was investigated using human whole cell extracts. The efficiency of rejoining of SSBs induced by Al(K) ultrasoft X rays is less than that observed for gamma-ray-induced SSBs. From the similarity of the extent of rejoining of SSBs induced by gamma rays under aerobic and anaerobic conditions, the chemical nature of the stand break termini does not significantly influence SSB rejoining. A simple nick induced in plasmid DNA by gpII protein is rejoined rapidly compared with the slower rejoining processes for radiation-induced SSBs. Therefore, ligation is not rate-determining in processing radiation-induced SSBs. This study provides further evidence that nonrejoining of radiation-induced SSBs reflects the complexity of DNA damage. From comparison of the extent of rejoining of SSBs induced by different radiations, it is inferred that double-strand breaks represent only a minor component of the overall yield of complex damage.     

Rectification of tandemly repetitious DNA. II. Clustering and periodicity

In a given segment of tandemly repetitious DNA, the repeating units are similar but not always identical. Current techniques in molecular biology allow determination of whether variants are clustered or randomly interspersed along the segment. Such findings have been used as evidence for, or against, different models for internal rearrangement of the DNA, such as the rolling circle model or unequal sister chromatid exchange. Information has not been available, however, as to the extent to which observed patterns of clustering or interspersion are compatible with the different models. In the present study, computer simulations were used to obtain this information. As measured by certain indices of clustering and periodicity, each model generates a reproducible pattern that is relatively independent of the degree of heterogeneity in the segment.     

Interactions of diastereomeric tripeptides of lysyl-5-fluorotryptophyllysine with DNA. 1. Optical and 19F NMR studies of native DNA complexes

Lysyl-5-fluoro-L-tryptophyllysine and lysyl-5-fluoro-D-tryptophyllysine were synthesized, and their interactions with double-stranded DNA were investigated as a model for protein-nucleic acid interactions. The binding to DNA was studied by monitoring various 19F NMR parameters, the fluorescence, and the optical absorbance in thermal denaturation. The 19F resonance of the L-Trp peptide shifts upfield in the presence of DNA, and that of the D-Trp peptide shifts downfield with DNA present. The influence of ionic strength on the binding of each peptide to DNA and the fluorescence quenching titration of each with DNA indicate that electrostatic bonding (approximately 2 per peptide-DNA complex) dominates the binding in each case and accounts for the similar binding constants determined from the fluorescence quenching, i.e., 7.7 X 10(4) M-1 for the L-Trp complex and 6.2 X 10(-1) for the D-Trp complex. The 19F NMR chemical shift, line width, 19F[1H] nuclear Overhauser effect, and spin-lattice relaxation time (T1) changes all indicate that the aromatic moiety of the L-Trp complex, but not that of the D-Trp complex, is stacked between the bases of DNA. The relative increases in DNA melting temperature caused by binding of the tripeptide diastereomers are also consistent with stacking in the case of the L-Trp peptide. The magnitude of the changes and the susceptibility of the 19F NMR chemical shift to altering the solvent isotope (H2O vs. D2O) suggest that the L-Trp ring is not intercalated in the classical sense but is partially inserted between the bases of one strand of the double helix.     

Isolation and characterization of calmodulin genes from Xenopus laevis.

Two cDNAs derived from Xenopus laevis calmodulin mRNA have been cloned. Both cDNAs contain the complete protein-coding region and various lengths of untranslated segments. The two cDNAs encode an identical protein but differ from each other by 5% nucleotide substitutions. The 5' and 3' untranslated regions, to the extent available, are highly homologous between the two cDNAs. The predicted sequence of X. laevis calmodulin is identical to that of vertebrate calmodulins from mammals and chickens and shows one substitution compared with electric eel calmodulin. Genomic DNA sequences homologous to each of the two cDNA clones have been isolated and were shown to account for the major calmodulin-coding DNA sequences in X. laevis. These data suggest that X. laevis carries two active, nonallelic calmodulin genes. Although no complete analysis has been carried out, it appears that the X. laevis calmodulin genes are interrupted by at least four introns. The relative concentrations of calmodulin mRNA have been estimated in different embryonic stages and adult tissues and found to vary by up to a factor of 10. The highest levels of calmodulin mRNA were found in ovaries, testes, and brains. In these three tissues, the two calmodulin genes appear to be expressed at approximately equal levels.     

The pattern of sex chromosome kinetochore phosphorylation during nonrandom segregation in a flea beetle.

In the flea beetle species, Alagoasa bicolor, males have two sex chromosomes, X and Y, each of which is larger than the rest of the genome combined. These large sex chromosomes do not pair at meiosis I, and are therefore not joined at metaphase I. Nevertheless, they always segregate from each other at anaphase I. As prometaphase I progresses, the unpaired X and Y undergo reorientation from a parallel to a linear configuration. Using 3F3/2, an antibody that detects the level of phosphorylation of a kinetochore protein or proteins, we have determined that this reorientation is not accompanied by a change in the level of phosphorylation of the kinetochores of either X or Y. This implies that: i) either the reorientation does not involve the loss or gain of kinetochore microtubules, or ii) if such loss or gain occurs, it does not effect a change in the tension placed on the nonrandomly segregating kinetochores, or iii) the sex chromosomes, as in some other species, have lost the ability to sense kinetochore tension changes. Evolution of nonrandom segregation may necessitate the inability of the participating chromosomes to affect the metaphase checkpoint.     

The interactions of haem with ligandin and aminoazo-dye-binding protein A.

1. The interactions of ferriprotoporphyrin IX with ligandin and aminoazo-dye-binding protein A result in absorption spectra in the Soret region characteristic of the ligand in its monomeric state. 2. Both proteins are able to bind ferrous as well as ferric haem. 3. Ferriprotoporphyrin IX is bound at a single site on both proteins. At pH7.0, I 0.16M, difference-spectrophotometric measurements gave association constants of 10(7) and 4 X 10(6) LITRE/MOL FOR LIGANDIN AND PROTEin A respectively. Under the same conditions fluorescence-quenching experiments gave an association constant of 2 X 10(7) litre/mol for ligandin. 4. Bilirubin, bromosulphophthalein and oesterone sulphate each compete with haem for binding by the two proteins. 5. Ferriprotoporphyrin IX bound to both ligandin and protein A is able to form co-ordination complexes with CN-, but not, to any measurable extent, with either N3- or F-. From these results it is suggested that binding by the two proteins may not involve the haem iron atom. 6. Both haem-protein complexes give rise to measurable extrinsic Cotton effects in the Soret region. 7. The formation and properties of the ligandin- and protein A-haem complexes are compared with those of haem-albumin, haemoglobin, myoglobin and other haemoproteins.     

Ultrastructure and behavior of the achiasmatic,telosynaptic XY pair of the sand rat (Psammomys obesus)

The behavior of the X and Y chromosomes in somatic and testicular cells of the sand rat (P. obesus) has been investigated with light and electron-microscope procedures. The Y chromosome has been identified as the fourth longest of the complement, both by C-banding and by its meiotic behavior. The X chromosome is the longest of the complement and carries two major C-heterochromatic blocks, one in the distal part of the long arm and the other forming most of the short arm. During presynaptic stages in spermatocytes, separate C-heterochromatic blocks, representing the sex chromosomes, are observed in the nuclei. An XY body is regularly formed at pachytene. During first meiotic metaphase the X and Y chromosomes show variable associations, none of them chiasmatic. Second meiotic metaphases contain, as in other mammals, a single sex chromosome, suggesting normal segregation between the X and the Y. — Electron microscopic observations of the autosomal synaptonemal complexes (SCs) and the single axes of the X and Y chromosomes during pachytene permit accurate, statistically significant identification of each of the largest chromosomes of the complement and determination of the mean arm ratios of the X and Y axes. The X and Y axes always lie close to each other but do not form a SC. The ends of the X and Y axes are attached to the nuclear envelope and associate with each other in variable ways, both autologously (X with X or Y with Y) and heterologously (X with Y), with a tendency to form a maximum number (four) of associated ends. Analysis of 36 XY pairs showed no significant preference for any single specific attachment between arm ends. The eighth longest autosomal bivalent is frequently partially asynaptic during early pachytene, and only at that time is often near or touching one end of the X axis. — It is concluded that while axis formation and migration of the axes along the plane of the nuclear envelope proceed normally in the X and Y chromosomes, true synapsis (with SC formation) does not occur because the pairing region of the X chromosome has probably been relocated far from the chromosome termini by the insertion of distal C-heterochromatic blocks.     

Differences among human immunodeficiency virus strains in their capacities to induce cytolysis or persistent infection of a lymphoblastoid cell line immortalized by Epstein-Barr virus.

Four strains of human immunodeficiency virus (HIV) manifest consistent differences in biologic behavior after infection of the X50-7 line of human umbilical cord lymphocytes immortalized by Epstein-Barr virus (EBV). Some dilutions of the first strain examined, human T-cell lymphotropic virus type III B, which is derived from a pool of patient isolates propagated in H9 cells, caused transient cytopathic effects (CPE) followed by recovery of a subpopulation of X50-7 cells which became virus carrier cultures. Other dilutions of the same virus stock completely lysed X50-7 cells. Two other strains, RF2 and YW, both from individual patients with acquired immune deficiency syndrome, always induced complete cytolysis of X50-7 cells at all dilutions which infected the cells. However, RF2 did establish persistent infection of H9 cells. A fourth strain, PH1-MN, from a child with acquired immune deficiency syndrome-related complex, induced only transient CPE in X50-7 and H9 cells, which thereafter always recovered to form carrier cultures. For all four strains, the dilutions of HIV stocks which caused CPE corresponded to dilutions which resulted in the detection of HIV polypeptides by immunoblot. Cytolysis in HIV-infected X50-7 cells was accompanied by a decrease in the amount of EBV nuclear antigen; however, HIV infection did not induce EBV replication. Thus CPE in X50-7 cells is due to replication of HIV per se and not to activation of EBV. The observations indicate that there are differences in the cytolytic properties of HIVs and that these differences are influenced by the target cell.     

Studies of the temporal relationship between the cytogenetic and biochemical manifestations of X-chromosome inactivation during the differentiation of LT-1 teratocarcinoma stem cells

Previous biochemical studies have suggested that both X chromosomes produce gene products when cells of the LT-1 teratocarcinoma stem cell line are maintained in the undifferentiated state, and that dosage compensation, the biochemical manifestation of X inactivation, occurs when the cells are induced to differentiate in vitro (Martin et al., 1978). In this study the differentiation of LT-1 cells in vitro is described in detail, and data from cytogenetic studies of the time of X-chromosome replication in LT-1 cells are presented. They show that as long as the cells are maintained in the undifferentiated state both X chromosomes in each cell show the isocyclic replication pattern typical of a genetically active chromosome. However, when the LT-1 cells are induced to differentiate under appropriate conditions, one of the two X chromosomes in each cell of a large proportion of the population displays the allocyclic (either early or late) replication pattern typical of an inactive X chromosome. These data thus confirm that undifferentiated LT-1 cells contain two active X chromosomes and that X inactivation occurs in differentiating cultures of LT-1 cells. It is further demonstrated that there is a close temporal correlation between the biochemical and cytogenetic manifestations of the X-inactivation process. In addition, we observed that although X inactivation does not occur in the absence of morphological differentiation, it does not always occur when the cells differentiate in vitro.     

Phosphorylation of the SQ H2A.X motif is required for proper meiosis and mitosis in Tetrahymena thermophila

Phosphorylation of the C terminus SQ motif that defines H2A.X variants is required for efficient DNA double-strand break (DSB) repair in diverse organisms but has not been studied in ciliated protozoa. Tetrahymena H2A.X is one of two similarly expressed major H2As, thereby differing both from mammals, where H2A.X is a quantitatively minor component, and from Saccharomyces cerevisiae where it is the only type of major H2A. Tetrahymena H2A.X is phosphorylated in the SQ motif in both the mitotic micronucleus and the amitotic macronucleus in response to DSBs induced by chemical agents and in the micronucleus during prophase of meiosis, which occurs in the absence of a synaptonemal complex. H2A.X is phosphorylated when programmed DNA rearrangements occur in developing macronuclei, as for immunoglobulin gene rearrangements in mammals, but not during the DNA fragmentation that accompanies breakdown of the parental macronucleus during conjugation, correcting the previous interpretation that this process is apoptosis-like. Using strains containing a mutated (S134A) SQ motif, we demonstrate that phosphorylation of this motif is important for Tetrahymena cells to recover from exogenous DNA damage and is required for normal micronuclear meiosis and mitosis and, to a lesser extent, for normal amitotic macronuclear division; its absence, while not lethal, leads to the accumulation of DSBs in both micro- and macronuclei. These results demonstrate multiple roles of H2A.X phosphorylation in maintaining genomic integrity in different phases of the Tetrahymena life cycle.     

Frequent derepression of G6PD and HPRT on the marsupial inactive X chromosome associated with cell proliferation in vitro

X chromosome dosage compensation in Marsupials is like that in eutherian mammals except that the paternal X chromosome is always inactive, and silence of this chromosome is not well maintained. We previously showed that the unstable inactivation of the paternal G6PD allele is associated with the lack of DNA methylation in the 5' CpG cluster. Even though this CpG island is unmethylated, the paternal allele (marked by an enzyme variant) is at least partially and often severely repressed in most tissues of the opossum, so that factors other than methylation must inactivate the locus. Here we report that when cell cultures are established from these tissues, the silent G6PD locus is depressed. Although often complete, the extent of derepression differs among tissues and within different cell types in the same tissue, and is not accompanied by obvious changes in the pattern of chromosome replication. Studies of the HPRT locus in these cells show that the paternal HPRT allele also derepresses in cultured cells. These observations suggest that without DNA methylation to maintain the silence of the locus, tissue or cell-specific factors act to repress the silent locus, but are unable to maintain inactivity through cell division, or are lost as cells proliferate in culture.     

Internuclear transfer of genetic information in kar1-1/KAR1 heterokaryons in Saccharomyces cerevisiae.

Heterokaryons of Saccharomyces cerevisiae have been constructed utilizing the kar1-1 mutation, which prevents nuclear fusion during conjugation (J. Conde and G. Fink, Proc. Natl. Acad. Sci. U.S.A. 73:3651-3655, 1976). Each heterokaryon contained two haploid nuclei that were marked on several chromosomes. They segregated haploid progeny (cytoductants), most of which have the nuclear genotype of one or the other of the heterokaryon parents, but they occasionally segregated progeny having a recombinant genotype (exceptional cytoductants). Exceptional cytoductants receive the majority of their genome from one parent (the recipient) and a minority from the other (the donor). Transfer of two markers from the donor nucleus to the recipient is rarely coincident for markers located on different chromosomes but is nearly always coincident for those markers located on the same chromosome, suggesting that whole chromosomes are transferred from the donor nucleus to the recipient. In crosses of kar1-1 X KAR1 parents, either nucleus may act as a recipient or donor with equal probability. Recipient nuclei acquired 9 of the 10 chromosomes examined, with frequencies which were inversely correlated with the size of the chromosome. When a chromosome is acquired by the recipient nucleus, it either replaces its homolog or exists in a disomic condition. Haploid progeny emanating from kar1 X KAR1 crosses are frequently inviable. I tested whether this inviability might be the result of chromosome loss by donor nuclei. Viability of progeny from kar1 X KAR1 heterokaryons was improved when the parental nuclei were diploid to an extent consistent with the hypothesis, and diploid progeny which had become monosomic were recovered from these heterokaryons. The following sequence of events accounts for chromosome transfer in kar1 X KAR1 heterokaryons. After cell fusion, each nucleus in the heterokaryon has a probability of about 0.38 of losing one or more chromosomes. A nucleus sustaining such a loss can become a donor in a chromosome transfer event. If the other nucleus does not sustain a mortal chromosome loss, it can become a recipient in a transfer event. The chance of acquiring a chromosome lost by the donor is greater for smaller chromosomes than for larger ones and is about 0.05 for the average chromosome.     

Sex-specific viability, sex linkage and dominance in genomic imprinting

Genomic imprinting is a phenomenon by which the expression of an allele at a locus depends on the parent of origin. Two different two-locus evolutionary models are presented in which a second locus modifies the imprinting status of the primary locus, which is under differential selection in males and females. In the first model, a modifier allele that imprints the primary locus invades the population when the average dominance coefficient among females and males is >12 and selection is weak. The condition for invasion is always heavily contingent upon the extent of dominance. Imprinting is more likely in the sex experiencing weaker selection only under some parameter regimes, whereas imprinting by either sex is equally likely under other regimes. The second model shows that a modifier allele that induces imprinting will increase when imprinting has a direct selective advantage. The results are not qualitatively dependent on whether the modifier locus is autosomal or X linked.     

Binding of beryllium to nuclear acidic proteins.

In vitro beryllium (Be) binding to rat liver nuclei has been reassessed (KAss = 2.0 X 10(6) M: n = 17 nmol Be/mg protein). Be also binds to rat liver nucleoli (KAss approx. 4 X 10(6) M: n = 10 nmol Be/mg protein). Examination of rat liver chromatin fractionated on a hydroxyapatite column shows that Be does not bind to histone or to the non-histone protein eluted by 0.05 M sodium phosphate. Be is strongly bound to the non-histone proteins eluted by 0.2 M sodium phosphate (KAss = 1.1 X 10(6) M: n = 55 nmol Be/mg protein) and also to the same extent to the fraction containing DNA which is subsequently eluted from the column. Evidence is provided that the latter binding is not due to DNA. The fractions containing the Be-binding proteins also contain the proteins which are phosphorylated to the greater extent.     

VALVE AND BAND MORPHOLOGY OF SOME FRESHWATER DIATOMS. II. INTEGRATION OF VALVES AND BANDS IN NAVICULA CONFERVACEA VAR. CONFERVACEA12

Chemically cleaned and critical-point dried cells of a clonal culture were examined with scanning electron microscopy. Cells form filaments by valve-to-valve connections maintained by organic material which adheres to the central area of the valve face. Bending of filaments is probably restricted to some extent by the articulation of overlapping spatulate marginal spines with an adjacent underlapping set of much shorter spines (ridges), and with the mantle edge itself. Cell division results in three possible spine patterns for each cell: a set of overlapping and a set of underlapping spines; no overlapping sets of spines (two underlapping); or two sets of overlapping spines (no underlapping). Each filament inherits cells with spine set patterns in the ratio of 2 (with 1 set overlapping): 1 (with no sets overlapping): 1 (with 2 sets overlapping). Valvocopulae are shaped similarly to pleurae except that the partes exteriores of the valvocopulae are wider. The pars interior of both is delimited by an advalvar row of pores continuous around the cell apex. The pars exterior also has a row of pores, but it is median in the valvocopula and first pleura and does not continue around the cell apex. The valvocopulae always underlap the mantle and the pleurae always underlap their preceding band. The ends of both appeared attached, but may become free in acid-cleaned preparations. Bands alternate with each other so that the ends of the valvocopula attach to the first continuous apical portion of the first pleura; the ends of the first pleura attach in that same fashion to the second pleura but at the opposite apex; and all subsequent pleurae alternate in the same fashion with up to at least 13 pleurae/epicingulum. The continuous apical portion of each band is elevated so that a functional (but not structural) ligula is formed, with the continuous apical portion of alternate bands becoming adjacent and underlapping each other only in this region. The valvocopulae in a single cell, or of adjacent cells, may have their continuous apical ends on the same or on opposite apices. It is recommended that N. confervacea var. peregrina (W. Sm.) Grun. be merged with the nominate variety.     

Survival of Escherichia coli in lake bottom sediment.

The survival of Escherichia coli in bottom sediment (Lake Onalaska, navigation pool no. 7, Mississippi River) was studied by using in situ dialysis culture of sterile (autoclaved) and unsterile sediment samples. Bags made from dialysis tubing were filled with either course sand sediment (28.8% fine) or organic, silty clay sediment (77.2% fine) and placed at the sediment-water interface. Bags representing sterile controls, unsterile uninoculated controls, autoclaved inoculated sediment, and unsterile inoculated sediment were studied during a 5-day period for each sediment type. Daily most-probable-number determinations indicated that E. coli populations in unsterile inoculated sediment fluctuated between 5.3 X 10(2) and 2.2 X 10(3) bacteria per g of silty clay and between 3.0 X 10(3) and 1.4 X 10(4) bacteria per g of sand. Autoclaved silty clay sediment inoculated with 1.0 X 10(6) bacteria per g increased to 2.2 X 10(8) bacteria per g in 3 days. During the same period, autoclaved sand sediment inoculated with 1.2 X 10(5) cells per g increased to 5.4 X 10(7) bacteria per g. By day 5, populations in both cultures had decreased by 1 log. The ability of E. coli to survive for several days in aquatic sediment in situ suggests that fecal coliforms in water may not always indicate recent fecal contamination of that water but rather resuspension of viable sediment-bound bacteria.