Electronic structure of dinuclear gold(i) complexes.

Cyclic voltammetry (CV) experiments on LL(AuSR *)(2) complexes [LL = diphenylphosphinomethane (dppm), diphenylphosphinopentane (dpppn); R(*) = p-SC(6)H(4)CH(3)] show anodic sweeps that broaden by about 25 mV on going from the longer (dpppn) to the shorter (dppm) bidentate phosphine ligand. Changing concentrations had no effect on the shape of the waveform. The result suggests a weak intramolecular metal-metal interaction in dppm(AuSR *)(2) that correlates well with rate acceleration occurring in the reaction of dppm(AuSR *)(2) with organic disulfides. Quantum yields for cis-dppee(AuX)(2) [dppee = 1,2-bis(diphenylphosphino)ethylene; X = Cl, Br, I] complexes, (disappearance) Phi , are significantly higher in complexes with a softer X ligand, a trend that correlates well with aurophilicity. This result also suggests that electronic perturbation caused by Au(I)-Au(I) interactions is important in explaining the reactivity of some dinuclear gold(I) complexes. The crystal structure for cis-dppee(Aul)(2) shows short intramolecular Au(I)-Au(I) interactions of 2.9526 (6) A degrees , while the structure of trans-dppee(AuI)(2) , shows intermolecular Au(I)-Au(I) interactions of 3.2292 (9) A degrees . The substitution of .As for P results in a ligand, cis-diphenylarsinoethylene (cis-dpaee), that is photochemically active, in contrast to the cis-dppee ligand. The complexes, cis-dpaee(AuX)(2), are also photochemically active but with lower quantum yields than the cis-dppee(AuX)(2) complexes.

Cloning, sequence and expression of a linear plasmid-based and a chromosomal homolog of chloroacetaldehyde dehydrogenase-encoding genes in Xanthobacter autotrophicus GJ10.

The degradation of 1,2-dichloroethane (DCE) by Xanthobacter autotrophicus GJ10 proceeds via chloroacetaldehyde (CAA), a toxic intermediate in the cells if it is not metabolized further by the NAD(+)-dependent CAA dehydrogenases. Here, we describe the cloning, sequence and expression in Escherichia coli of aldA, a plasmid-located CAA dehydrogenase-encoding gene of GJ10 as well as a chromosomal homolog, designated aldB. The DNA-predicted amino acid (aa) sequences of the two proteins (505 aa in AldA and 506 aa in AldB) are 84% identical. The cloned aldA and aldB genes were verified by their expression in the E. coli T7 polymerase/promoter and the pUC lac promoter systems. The expression level of AldA and its enzymatic activity towards CAA were both higher than those of AldB. In a hybrid construct, the 3'end of aldB was able to complement, although not completely, the corresponding portion of aldA to produce a functional gene. Both AldA and AldB proteins of GJ10 share the highest degree of sequence identity with an acetaldehyde dehydrogenase (ALDH) encoded by acoD of Alcaligenes eutrophus (77.3-78% identity). Together with at least three other ALDHs of prokaryotic origin, these proteins apparently form a special class of ALDHs whose expressions are dependent on RpoN factors. By pulsed-field gel electrophoresis the 225-kb pXAU1 plasmid encoding aldA was shown to be linear.

Phototoxicity of some bromine-substituted rhodamine dyes: synthesis, photophysical properties and application as photosensitizers.

The synthesis of some bromine-substituted rhodamine derivatives viz., 4,5-dibromorhodamine methyl ester (dye 2) and 4,5-dibromorhodamine n-butyl ester (dye 3) are reported. These dyes were synthesized to promote a more efficient cancer cell photosensitizer for potential use in in vitro bone marrow purging in preparation for autologous bone marrow transplantation. Spectroscopic and photophysical characterization of these dyes together with rhodamine 123 (dye 1) are reported in water, methanol, ethanol and also in a microheterogeneous system, sodium dodecyl sulfate. The possible mechanism of photosensitization is characterized in terms of singlet oxygen efficiency of these dyes. Singlet oxygen quantum yields for bromine-substituted dyes are in the range of 0.3-0.5 depending on the solvent. For dye 1 no singlet oxygen production is found. The photodynamic actions of these dyes in different cell lines are tested. It was found that dye 2 and dye 3 are efficient photosensitizers and mediate eradication of K562, EM2, myeloid cell lines (CML) and the SMF-AI rhabdomyosarcoma line.

Preparation, manipulation, and pulse strategy for one-dimensional pulsed-field gel electrophoresis (ODPFGE).

The underlying principles for zero-integrated-field electrophoresis (ZIFE) pulses and more general forward-biased pulse schemes are reviewed for one-dimensional pulsed-field gel electrophoresis (ODPFGE) separations of large DNA molecules. Detailed descriptions of materials, preparation protocols, hardware requirements, and procedures are given. A variety of gel pictures for known yeast DNA markers are shown.

The Myb and Ahi-1 genes are physically very closely linked on mouse chromosome 10.

Ahi-1 has previously been identified as a common helper provirus integration site on mouse Chromosome (Chr) 10 in 16% of Abelson pre-B-cell lymphomas and shown to be closely linked to the Myb protooncogene. By using long-range restriction mapping, we have mapped the Myb and Ahi-1 regions within a 120-kbp DNA fragment. The Ahi-1 region is located approximately 35 kbp downstream of the Myb gene. A further confirmation of this finding was obtained by screening a mouse YAC library. The three positive clones obtained contained both the Myb and Ahi-1 gene sequences. To test whether provirus integration in the Ahi-1 region enhances the expression of Myb by a cis-acting mechanism, we have also examined Myb gene expression in A-MuLV-induced pre-B-lymphomas. Our data have revealed that there is no clear evidence for such activation in the tumors we have tested, indicating that provirus insertion in the Ahi-1 region is activating a novel gene, apparently involved in tumor formation.

Redox chemistry of gold(i) phosphine thiolates: sulfur-based oxidation.

The redox chemistry of mononuclear and dinuclear gold(I) phosphine arylthiolate complexes was recently investigated by using electrochemical, chemical, and photochemical techniques. We now report the redox chemistry of dinuclear gold(I) phosphine complexes containing aliphatic dithiolate ligands. These molecules differ from previously studied gold(I) phosphine thiolate complexes in that they are cyclic and contain aliphatic thiolates. Cyclic voltammetry experiments of Au(2) (LL)(pdt) [pdt = propanedithiol; LL = 1,2-bis(diphenylphosphino)-ethane (dppe), 1,3-bis(diphenylphosphino)propane (dppp), 1,4-bis(diphenylphosphino)butane (dppb), 1,5-bis(diphenylphosphino)pentane (dpppn)] in 0.1 M TBAH/CH(3)CN or CH(2)Cl(2) solutions at 50 to 500 mV/sec using glassy carbon or platinum electrodes, show two irreversible anodic processes at ca. +0.6 and +1.1 V (vs. SCE). Bulk electrolyses at +0.9 V and +1.4 V result in n values of 0.95 and 3.7, respectively. Chemical oxidation of Au(2)(dppp)(pdt) using one equivalent of Br(2) (2 oxidizing equivalents) yields 1,2-dithiolane and Au(2)(dppp)Br(2). The reactivity seen upon mild oxidation /= +1.3 V) is consistent with oxidation of gold(I) to gold(III). Structural and electrochemical differences between gold(I) aromatic and aliphatic thiolate oxidation processes are discussed.

Long-range mapping of Mis-2, a common provirus integration site identified in murine leukemia virus-induced thymomas and located 160 kilobase pairs downstream of Myb.

The nondefective Moloney murine leukemia virus (MuLV) induces clonal or oligoclonal T-cell tumors in mice or rats. The proviruses of these nondefective MuLVs have been shown to act as insertion mutagens most frequently activating an adjacent cellular gene involved in cell growth control. Mutations by provirus insertions, recognized as common provirus integration sites, have been instrumental in identifying novel cellular genes involved in tumor formation. We have searched for new common provirus integration sites in Moloney MuLV-induced thymomas. Using cellular sequences flanking a provirus cloned from one of these tumors, we found one region, designated Mis-2, which was the target of provirus integration in a low (3%) percentage of these tumors. Mis-2 was mapped on mouse chromosome 10, approximately 160 kbp downstream of myb. The Mis-2 region may contain a novel gene involved in tumor development.

Effect of one-dimensional pulsed-field gel electrophoresis on linear and circular DNA.

A summary of the three main one-dimensional pulsed-field strategies (zero-integrated field, forward-biased field, and high frequency modulation) used for separating DNA molecules without band inversion within a preselected size range is given. Each of these strategies has size-specific features which make separations up to 6 Mbp possible. We applied the same methodology to circular DNAs varying in size from 2 kbp to about 4 Mbp. The migration of intermediate-sized circular plasmids (50 kbp-400 kbp) under these pulse conditions remains unexplained. On the other hand, preliminary results show that the migration of very large molecules, which are expected to be circular, comigrate with linear chromosomes of the same size under certain pulse conditions. We hypothesize that sample preparation, or the effect of the pulsed field, can create breakage and linearize very large circular DNAs, or that very large circular DNAs (> 2 Mbp) act like linear DNAs of the same size when submitted to one-dimensional pulsed-field gel electrophoresis conditions. The most likely possibility is that some of the circular DNAs have been linearized with one break during sample preparation, giving rise to a band at about 4 Mbp. The circular DNAs with more than one break may form an indistinguishable smear.

Manual sequencing using pulsed field.

The use of pulsed fields in manual sequencing opens up the compression zone found with a DC field and extends the range of resolution from a few hundred bases to several thousand bases. The band inversion problem is overcome with the proper pulsing conditions, and the bands are sharper than for the DC field case. Accurate visual reading is possible up to about 800-900 bases. The method is compatible with automation techniques, since the band spectrum is stretched continuously during migration, and the smaller fragments are run off the gel.

Pulsed field sequencing gel electrophoresis.

The effect of pulsed fields on sequencing gel electrophoresis is investigated, using DNA fragment markers ranging in size from 20 to 6557 bases. For high continuous electric fields (5000 V/55 cm) band inversion is observed in which fragments larger than 4000 bases migrate faster than those of 800-1000 bases. The use of one-dimensional pulsed field gel electrophoresis (ODPFGE) eliminates band inversion and extends the monotonic size-mobility relationship of the DNA markers up to about 4000 bases. The relevance of these results, obtained using a manual sequencing process with autoradiographic detection, to automated sequences is discussed.

Preparation, Manipulation, and Pulse Strategy for One-Dimensional Pulsed-Field Gel Electrophoresis (ODPFGE).

The preparation and manipulation of very large DNA molecules for pulsed-field gel electrophoresis (PFGE) requires more care than normally used for smaller molecules. The general protocol used is the preparation of DNA directly in solid agarose blocks (plugs) or beads. Intact cells are encapsidated in agarose (plugs or beads) and are treated with different combinations of enzymes and detergent to remove cell walls, membranes, RNA, proteins, and other materials in order to obtain naked DNA. This is possible because detergent and enzymes can diffuse by Brownian motion through the agarose pores of the plug, whereas the large pieces of DNA cannot and remain sequestered inside the plug. Here, we give detailed protocols of preparation for yeast and mammalian DNA. In addition, we describe the power supplies, switchers, gels, and gel trays required for one-dimensional pulsed-field gel electrophoresis (ODPFGE), as well as the pulse strategies used for the separations.

Preparation and Separation of Intact Chromosomes of Vertebrates by One-Dimensional Pulsed-Field Gel Electrophoresis (ODPFGE).

This novel way of preparing chromosomes for pulsed-field gel electrophoresis (PFGE) takes advantage of the fact that the whole chromosome population is synchronized in metaphase. This is a very important step toward their intact separation by PFGE; for instance, a standard preparation as used for digestion with rarecutter enzymes shows a different pattern of resolution, characterized by diffuse bands and nonspecific migration (see Chapter 7 ). Here, vertebrate chromosomal DNA was prepared by a modified chromosome isolation procedure for flow cytometry (1). This procedure involves lysis of cells (blocked in metaphase by colcemide) with digitonine in the presence of spermidine and spermine as described below. The structural integrity of metaphase-blocked chromosomes is given by the presence of spermine and spermidine, which act as chromosomal morphology stabilizers. The digestion with proteinase K is carried out as described before in order to eliminate chromosomal proteins. The pulse parameters for separating intact chicken microchromosomes by one-dimensional pulsed-field gel electrophoresis (ODPFGE) are also given.

Elastic Bag Model of One-Dimensional Pulsed-Field Gel Electrophoresis (ODPFGE).

Gel electrophoresis is one of the most common techniques used in molecular biology for the separation of DNA molecules. Conventional gel electrophoresis (using a static electric field) does not permit separation of DNA fragments larger than 30-50 kbp (1) as shown in Fig. 1A of Chapter 7 . This is a surprising result as one would think that larger molecules would suffer a larger retardation, and separation over any size range would be possible. The inability to separate is related to the molecular conformation of a polyelectrolyte, such as DNA, migrating in a disordered medium, such as a gel, under the influence of a static electric field. During continuous field electrophoresis, the larger DNA fragments tend to orient and stretch in the field direction because they migrate in a one-dimensional fashion between the gel fibers (2-4). When this orientation is negligible, e.g., for smaller molecules or for very low field intensities, they maintain a three-dimensional random-walk conformation intertwined with the gel fibers during migration, and experience a retardation that is proportional to the mol size. However, when the orientation becomes large, the molecules become stretched and migrate essentially linearly along the field direction (5). The electrophoretic mobility then becomes independent of the mol size and no separation of molecules of different sizes is possible (Fig. 1A of Chapter 7 ). Physically, this is a consequence of the fact that for long molecules stretched and oriented in the field direction, both the electrical force on the molecule and the average friction opposing the forward motion are proportional to the length. It follows that the velocity, which is the ratio of these two quantities, depends only on the force per unit length and is independent of the actual mol length. This explains why a plateau of length-independent mobility is reached in a continuous electric field (Fig. 2 of Chapter 7 ).

Observation of orientation and relaxation of protein-sodium dodecyl sulfate complexes during pulsed intermittent field polyacrylamide gel electrophoresis.

Polyacrylamide gel electrophoresis (PAGE) of proteins denatured with SDS (sodium dodecyl sulfate) has been used successfully to separate proteins according to their molecular mass. In spite of the extensive use of this technique, the motion of the protein-SDS complex in a polyacrylamide gel is still not understood. Here we report on the observation of the orientation (in the field direction) and relaxation of protein-SDS complexes during pulsed intermittent field PAGE experiments. The results give an indication of the stiffness of the molecules and may be useful for the development of a technique to improve the separation of large proteins using pulsed electric fields.

Construction of the physical map for three loci in chromosome band 13q14: comparison to the genetic map.

Pulsed-field gel electrophoresis (PFGE) and deletion mapping are being used to construct a physical map of the long arm of human chromosome 13. The present study reports a 2700-kilobase (kb) Not I long-range restriction map encompassing the 13q14-specific loci D13S10, D13S21, and D13S22, which are detected by the cloned DNA markers p7D2, pG24E2.4, and pG14E1.9, respectively. Analysis of a panel of seven cell lines that showed differential methylation at a Not I site between D13S10 and D13S21 proved physical linkage of the two loci to the same 875-kb Not I fragment. D13S22 mapped to a different Not I fragment, precluding the possibility that D13S22 is located between D13S10 and D13S21. PFGE analysis of Not I partial digests placed the 1850-kb Not I fragment containing D13S22 immediately adjacent to the 875-kb fragment containing the other two loci. The proximal rearrangement breakpoint in a cell line carrying a del13(q14.1q21.2) was detected by D13S21 but not by D13S10, demonstrating that D13S21 lies proximal to D13S10. Quantitative analysis of hybridization signals of the three DNA probes to DNA from the same cell line indicated that only D13S10 was deleted, establishing the order of these loci to be cen-D13S22-D13S21-D13S10-tel. Surprisingly, this order was estimated to be 35,000 times less likely than that favored by genetic linkage analysis.

Molecular detrapping and band narrowing with high frequency modulation of pulsed field electrophoresis.

In high electric fields, megabase DNA fragments are found to be trapped, i.e. to enter or migrate in the gel only very slowly, if at all, leading to very broad electrophoretic bands and loss of separation. As a consequence, low electric fields are usually used to separate these molecules by pulsed field electrophoretic methods. We report here that high-frequency pulses eliminate the molecular trapping found in continuous fields. When high frequency pulses are used to modulate the longer pulses used in pulsed field electrophoresis, narrower bands result, and higher fields can be used. We suggest that this is due to effects that occur on the length scale of a single pore.

DNA gel electrophoresis: effect of field intensity and agarose concentration on band inversion.

We study the effect of electric field intensity and agarose gel concentration on the anomalous electrophoretic mobility recently predicted by the biased reptation model and experimentally observed for linear DNA fragments electrophoresed in continuous electric fields. We show that high fields and low agarose concentrations eliminate the physical mechanism responsible for anomalous DNA mobility and band inversion, in good agreement with theory, thus restoring the monotonic mobility-size relationship necessary for unambiguous interpretation of the results of DNA gel electrophoresis.

Development and characterization of continuous avian cell lines depleted of mitochondrial DNA.

Populations of quail and chicken cells were treated with ethidium bromide, an inhibitor of mitochondrial DNA replication. After long-term exposure to the drug, the cell populations were transferred to ethidium bromide (EtdBr)-free medium, and cloned. Clones HCF7 (quail) and DUS-3 (chicken) were propagated for more than a year, and then characterized. Analysis of total cellular DNA extracted from these cells revealed no characteristic mitochondrial DNA molecule by Southern blot hybridization of HindIII- or AvaI-digested total cellular DNA probed with cloned mitochondrial DNA fragments. Reconstruction experiments, where a small number of parental cells was mixed with HCF7 cells and DUS-3 cells before extraction of total cellular DNA, further strengthen the notion that the drug-treated cells are devoid of mitochondrial DNA molecules. The cell populations were found to proliferate at a moderately reduced growth rate as compared to their respective parents, to be auxotrophic for uridine, and to be stably resistant to the growth inhibitory effect of EtdBr and chloramphenicol. At the ultrastructural level, mitochondria were considerably enlarged and there was a severe reduction in the number of cristae within the organelles and loss of cristae orientation. Morphometric analysis revealed a fourfold increase of the mitochondrial profile area along with a twofold decrease of the numerical mitochondrial profiles. Analysis of biochemical parameters indicated that the cells grew with mitochondria devoid of a functional respiratory chain. The activity of the mitochondrial enzyme dihydroorotate dehydrogenase was decreased by 95% and presumably accounted for uridine auxotrophy.

Scrambling of bands in gel electrophoresis of DNA.

Under certain conditions of agarose gel electrophoresis, larger DNA molecules migrate faster than smaller ones. This anomalous mobility of DNA, which can lead to serious errors in the measurement of DNA fragment lengths, is related to near-zero velocity conformations which can trap DNA chains during electrophoresis. Intermittent electric fields can be used to alter the chain conformations so as to restore the monotonic mobility-size relationship which is necessary for a correct interpretation of the gel. These data are in agreement with the results of a computer simulation based on a theoretical model of electrophoresis.