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One difficulty in studying molecular changes of tumours has been the inability to isolate DNA and RNA from a homogeneous cell population. The combination of several new technologies should help overcome these hurdles. Microdissection is a technique for rapid and easy procurement of a pure cellular subpopulation away from its complex tissue milieu. Laser-assisted microdissection has recently been identified as a quick, simple and effective method by which microdissection of complex tissue specimens can be routinely performed for molecular analysis. With the advent of laser microdissection, cDNA libraries can be developed from pure cells obtained directly from stained neoplastic tissue, and microarrays of thousands of genes can now be used to examine gene expression in microdissected tumour tissue samples. This review will concentrate on the application of different microdissection techniques in the area of cancer research.

Detailed studies of chromosome rearrangements within solid tumors require karyotype analysis after cell culturing. However, different cell subpopulations with various growth capacities within one tumor may introduce biases in karyotype analysis, known as the in vitro selection. In our laboratory, 22% of karyotypes from breast cancers established after short-term culture were normal. Using interphase fluorescence in situ hybridization (FISH) for the determination of chromosome 1 arm imbalances and flow cytometry measurements of ploidy, we demonstrated that at least 2/3 of these tumors were mainly composed of aneuploid cell populations. Thus, the incidence of normal or balanced karyotypes among breast cancers is probably below 7%. This is the first direct proof for the existence of an in vitro selection within breast cancer cultures, suggesting cautious interpretation of cytogenetic data.

Interphase cytogenetics have become a widespread tool for investigation of chromosome rearrangements in solid tumors. The most recurrent chromosome alteration within breast cancer affects chromosome 1, leading principally to gain of the long arm and/or loss of the short arm. We have developed a new method for detection of chromosome 1 arm imbalances in interphase nuclei. The method is based on quantitation of the fluorescence signals emitted by the hybridized two-color paintings of the short and long arms using image cytometry. The chromosome arm imbalance was determined by calculating the ratio of both fluorescence emissions of each arm. The ratio of the paintings of normal lymphocytes was used as a reference. Three breast cancer cell lines, 13 fresh tumor samples, and 6 fine-needle samplings of breast cancer were analyzed using an automated image cytometer. Whenever possible, classic cytogenetics and in situ hybridization on metaphases were performed as controls. Fluorescence ratios representing the imbalances of chromosome 1 arms with values between 1 and 3.2 were measured. Data between classic cytogenetics and interphase cytogenetics were well-correlated (r = 0.89). This method, which enables an easy detection of intrachromosomal imbalances without need of metaphase preparations, detects malignant cells and can be extended to other carcinomas for which chromosome 1 arm imbalances are recurrent or chromosome alterations specific of other malignancies. In comparison to other interphase fluorescence in situ hybridization techniques, it avoids every spot scoring problem encountered when using centromeric probes and the difficulties in interpreting structural rearrangements.

Angiogenesis is a complex phenomenon likely to be under the strict control of a group of transcription factor(s). Homeobox (HOX)-containing proteins have been identified as regulators controlling the coordinated expression of genes involved in organ development and tissue differentiation. In this study, we have demonstrated that human umbilical vein endothelial cells (HUVEC) express 8 of the 10 HOX genes contained in cluster B. Treatment of HUVEC with tissue plasminogen activator (TPA), an agent known to induce morphologic changes in endothelial cells, or vascular endothelium growth factor (VEGF), a proliferative and angiogenesis inducer, results in a specific time-dependent modulation of the eight HOX genes identified. Interestingly, neither basic fibroblast growth factor, an endothelial proliferative agent, nor TNP-470, a fumagillin derivative with potent antiendothelial cell proliferation properties, affected expression of these HOX genes. Specific modulation of HOX genes by differentiating agents but not by proliferative or antiproliferative molecules suggests that they could be involved in the control of the genetic program that coordinates the construction of new blood vessels.

DNA methylation is known to be related to the regulation of gene expression. DNA methylation patterns are modified in tumors compared to normal tissue, and in some cases, these variations are linked to cancer progression. Molecular biology techniques are generally used to evaluate DNA methylation status. We describe here a simple and fast immunofluorescent method to quantitate in situ DNA methylation using an image analyser and a CCD camera. Quantification of relative methylation levels in interphase nuclei and metaphase chromosomes was performed on digital images of two types of cells with known methylation levels. The results from image cytometry corresponded to those obtained by molecular studies. Advantages of this approach are that all the DNA of a cell may be examined rather than a limited restriction sequence, and that it may be done on a cell by cell basis rather than on a heterogenous population. In addition, with this method, changes in methylation patterns during tumor progression could be followed and eventually used as a marker for prognosis.

A procedure including incorporation of 5-bromodeoxyuridine (BrdU) in DNA and a thermal denaturation step was developed to obtain both R-banding and efficient binding of anti-5-methylcytosine antibodies on metaphase chromosomes. BrdU incorporation improved the efficiency of antibody binding disclosed by immunofluorescence staining. This method allowed semiquantitative analysis of the antibody binding sites on straightforward characterized metaphase chromosomes and was applied to normal human lymphocytes and lymphoblastoid cell lines for which DNA methylation status had been previously analyzed. A correlation was established between level of DNA methylation and the semiquantitative estimate of antibody fixation. This procedure can be used to study DNA methylation on metaphase chromosomes in transformed and cancerous cell lines.

Nine cases of malignant gliomas were selected for the presence of double minutes (dmin) or homogeneously staining regions (hsr) detected by conventional cytogenetics. Analyses were performed on fresh (2 cases) or xenografted (5 cases) tumors or both (2 cases). A modified comparative genomic hybridization technique (mCGH) was applied exhibiting a single amplified locus in 8 tumors and 4 amplified loci in one tumor. Recurrent sites of amplification were detected in 7p11-p12 (5 cases) and 1q32.1 (2 cases). Signals were also observed in 4q11-q12, 5p15.1, 7q31, 8q24.1 and 9p2 in one tumor each. Southern blotting demonstrated that the genes for EGFR (epidermal growth factor receptor), PDGFRA (platelet derived growth factor receptor alpha), MET and MYC oncogenes were involved in 7p11-p12, 4q11-q12, 7q31 and 8q24.1 amplifications, respectively. These amplifications were found by in situ hybridization on tumor spreads, in dmin or episomes for EGFR, dmin for PDGFRA and MET, and hsr and dmin for MYC genes. Other mCGH signals, for which no target genes could be proposed, were confirmed by chromosome paintings on tumor metaphases. In one of the tumors, the coamplification of DNA from 5p15.1 and 9p2 bands in the same dmin was demonstrated.

Current Topics in Microbiology and Immunology. Vol. 249: DNA Methylation and Cancer edited by P. A. Jones and P. K. Vogt Springer-Verlag (2000) pp. 170. ISBN 3–540-66608-7 75.50/$129.00 After a long period of relative confidentiality, the DNA methylation field has become a major research domain over the last few years. In this context, the importance of DNA methylation in human cancer has only become apparent over the last 5 to10 years. This small book (9 articles) provides a comprehensive overview of the main data and, more interestingly, presents the new concepts emerging from the recent extensive work, essentially performed over 2–3 years. The article written by B. Hendrich and A. Bird gives an overview of our current knowledge about the proteins implicated in DNA methylation, including DNA-methyltransferases and methylated-DNA-binding-proteins. It should be noted that the discovery of several of these proteins is a direct consequence of the human genome sequencing program, since they were first found ‘in silico’ by searching the databases. The specific properties of each of these partners of DNA methylation are beginning to be identified. Their implication in the regulation of histone acetylation suggests some possible mechanisms for regulation of gene expression. These models take into account, in particular, the remodeling of the chromatin structure. The value of mouse models in the understanding of the role of these proteins is discussed by P. W. Laird in another article. The present limitations of these approaches, essentially due to the non-viability of homozygous mutant mice for the main DNA-methyltransferase (Dnmt1) could be passed in the near future by the generation of conditional knockouts. Three articles by J. G. Herman and S. B. Baylin, M. F. Chan, G. Liang and P. A. Jones and J. P. Issa focus on the role of CpG island methylation in cancer and aging. These small stretches of DNA are frequently located around the transcription-start sites of approximately half of all human genes. For virtually all of these genes, with the exception of genes of the inactive X chromosome and some imprinted genes, these regions are maintained free of methylation in normal cells regardless of whether these genes are transcribed. It has been recognized that the CpG islands of a growing number of genes, either known to be involved in carcinogenesis (p16, E-cadherin, hMLH1,.) or candidate tumor supressor genes (p15, GST-Π,.) are methylated in many types of human cancer. The implication of the hypermethylation of CpG islands in tumor progression is discussed in its various aspects. In particular, the article by Chan et al. highlights the necessity to not oversimplify the relationships between methylation/inactivation and demethylation/activation. Moreover, extending his work on cancer, J. P. Issa shows that specific genes are affected by age-related methylation (EGFR, ER,.) and that such hypermethylation has disastrous consequences for the integrity of aged tissues. The article of A. P. Feinberg covers another area in this field and discusses the role of DNA methylation in imprinting and proposes a model for a role for the of loss of imprinting in cancer. Two articles investigate the action of tumor causing agents: the exogenous carcinogens and the Epstein-Barr virus (EBV). G. P. Pfeifer, M. S. Tang and M. F. Denissenko present the now well known effect of the deamination of methylcytosine on the formation of mutations. However, they insist on the finding that cytosine methylation can increase the rates of mutation by enhancing the binding of chemical carcinogens to DNA. This mechanisms is likely to have important implications for both chemical and ultra violet light induced carcinogenesis. K. D. Robertson summarize his work on the consequences of the inactivation of EBV genes on the virus' life cycle. The use of demethylating agents, like azacytidine, for reactivation of Cp-derived antigens, which could result in specific immune recognition of the tumor, is an interesting idea; however, as analyzed by M. (ABSTRACT TRUNCATED)

The glutathione S-transferase gene (GST pi) is located on the same chromosome band (11q13) as proto-oncogenes INT2 and HSTF1 which are frequently amplified in breast cancer. Using the Southern blot technique, we looked for the amplification of the GST pi gene in 17 fresh tumors from human mammary carcinoma. The tumors were preselected because either they had an amplification of the INT2 proto-oncogene detected by dot blot, or their karyotypes exhibited or did not exhibit homogeneously staining regions, a cytogenetic character indicating amplification. Coamplification of GST pi, HSTF1 and INT2 was observed in five tumors, and coamplification of GST pi and HSTF1 without amplification of INT2 in another tumor. We also observed coamplification of GST pi, INT2, HSTF1 in the mammary carcinoma cell line MDA/MB134, whereas GST pi alone was amplified in the mammary epithelial cell line HBL100. These results indicate that INT2, HSTF1 and GST pi belong to the same large amplicon. Since GST pi is involved in intracellular detoxication and since chemotherapeutic drugs are among its substrates, it will be of interest to study GST pi gene expression as well as the response to chemotherapy in patients presenting this amplicon.

Inactivation of both alleles of the retinoblastoma susceptibility gene ( RB1 ), localised on chromosome band 13q14, is the crucial event for the development of retinoblastoma, a malignant tumour that originates from embryonal retinal cells.1, 2 Germinal mutation of one allele leads to a predisposition to retinoblastoma. Tumour development is initiated by inactivation of the second allele. In most families, the tumour predisposition segregates as an autosomal dominant trait with high (90%) penetrance3 and affected subjects usually develop bilateral multifocal tumours. Rare instances of familial retinoblastoma with low penetrance and variable expressivity have been described. In such families, patients develop retinoblastoma later than classical familial cases, there is a high rate of unilateral cases, and phenotypically normal obligate carriers are observed.4 A family with several cases of retinoblastoma has been identified (fig 1). Four members of the family were affected; three had unilateral retinoblastoma (III.5, IV.12, and IV.13), diagnosed at 12, 12, and 24 months, respectively, and one had bilateral retinoblastoma (IV-6), which developed at 11 months. Thus, with the exception of patient IV.13, the age of onset was not delayed compared to an average of less than 12 months in the majority of familial retinoblastoma. There were no cases of spontaneous osteosarcoma in this family, a secondary tumour recurrently found in patients with predisposition to retinoblastoma.5 However, the retinoblastoma of patient IV.6 was treated by radiotherapy (45 Gy) and 10 years later an osteosarcoma occurred in the irradiated field. It is well established that a germline mutation in the RB1 gene dramatically increases the probability of developing a radiation induced osteosarcoma.6 After ophthalmic examination of unaffected family members, no case of retinoma/retinocytoma, which may represent benign proliferation or tumour regression, was found. Altogether, these observations suggest the presence of a germline RB1 mutation linked to incomplete …

The polarographic behaviour of DNA extracted from the calf thymus has been studied using phase sensitive a.c. polarography. The conditions of adsorption on the dropping mercury electrode have been studied in terms of the conditions of medium (ionic strength, pH) and of the molecular weight of DNA. The ionic strength plays a deciding role in the conditions of adsorption. The adsorption also depends on the form of the macromolecule. When it is in rod form (low molecular weight) the adsorption occurs parallel to the charged surface. When it adopts a wormlike form, loops are formed which extend into the solution. The adsorption does not depend on the nature of the monovalent counterion thus the adsorption is identical in Na + and NH 4 + media. The polarograms of native and denatured DNA present an initial rounded peak situated at the limit of the adsorption zone. The native DNA is characterised by a second peak which appears, whatever the medium and the molecular weight of the DNA may be, on the recordings of the in-phase component of the current. This peak is situated at a more positive potential than that which is normally characteristic of the denatured DNA. The characteristics of the peaks of the native DNA have been specified. The first peak corresponds to the desorption of a certain number of adsorbed elements of the macromolecule. The second peak changes its properties according to the pH. In a slightly acid medium, it corresponds to the reduction of the adsorbed bases without desorption of the reduced products. The zones of reduction are the locally destabilized, i.e. the A-T rich regions of the DNA. In alkaline medium it corresponds to the desorption of the macromolecule. A general schema of the behaviour of native DNA on the dropping mercury electrode is proposed.

The effects of ultrasonication treatment and of γ-irradiation damages on the molecular weight and the conformation of native calf thymus DNA have been elucidated by application of sweep voltammetry at a stationary mercury electrode (HMDE). This is a well established approach for the investigation of the hydrodynamic properties and the interfacial behaviour of DNA and other nucleotides at charged interfaces. Below pH 7, the faradaic response, connected with the reduction of protonated adenine and cytosine moieties from those zones of adsorbed native DNA where during the adsorption time t s before the sweep the double helix has opened can be utilized to monitor the sonication and radiation effects on DNA in a twofold manner depending on the fact whether full coverage of the interface with adsorbed DNA had been attained during the adsorption time t s before the sweep, or not. In the first case the reduction response is controlled by the diffusion rate of DNA from the bulk towards the interface electrode¦solution and the evaluated diffusion coefficient of the polynucleotide is correlated to its weight average molecular weight. For ultrasonication a good agreement between diffusion coefficients resulting from voltammetric and sedimentation measurements is observed. The result of sonication is merely a cutting of the native DNA-polymer into smaller analogous parts without affecting significantly its double

The electrochemical behaviour of DNA of various origin and of biosynthetic polynucleotides at a charged mercury/solution interface was studied in relation to their respective composition in bases. Two independent methods, phase sensitive a.c. polarography and single sweep voltammetry, at a hanging mercury drop electrode were used to ensure the reliability of the results. As all the biopolymers studied had a double-stranded conformation, the unwinding of these double-strand form due to the adsorption forces and to the electric field in the interface is indicated. The height of the reduction peak in single sweep voltammetry and its time integral vary linearly with the percentage of the bases A-T (adenine-thymine) in the biopolymer. The variation of the height and of the potential of a further capacitive a.c. peak shows that the phenomenon of reorganisation at a charged surface is also correlated to the nature of the bases. These results emphasize the importance of the base composition of the biopolymer as well as of its molecular weight and its conformation (single-stranded or double-stranded) in the course of niteraction with a charged surface.

The hydrolysis of DNA by the micrococcal nuclease results in the appearance of an a.c.-polarographic signal, the intensity of which can be directly related to the extent of the degradation. A thorough study of the behaviour of this signal, as a function of pH and ionic strength, evidences its essentially faradaic origin, associated with reductive phenomena involving the adenines and cytosines adjacent to the enzymatic breakages. For a constant ratio nuclease: DNA, the degradation rate depends on the origin and base composition of the DNA. Our observations suggest the existence of an initial specificity of the nuclease, in relation with its two possible mechanisms fo degradation.

All experimental results obtained in our laboratory during the last few years concerning the behaviour of various native nucleic acids, on a mercury | solution interface, are presented. On the basis of these results and taking into account the dynamic structure of DNA's as indicated by Von Hippel and Prinz, a general pattern of the behaviour of native DNA and of its polynucleotidic derivatives on a charged interface is proposed.

The conformational changes induced by negative supercoiling in the AC-rich region of the repeat unit of a Cebus satellite DNA has been studied by chemical probes sensitive to alterations in DNA conformation. This region is constituted of a (GT/CA)n stretch (15 less than or equal to n less than or equal to 18) associated to a sequence rich in GT/CA. At high superhelical density, at least 100 base pairs in the AC-rich region adopt the Z conformation as judged by diethyl pyrocarbonate reactivity. This is confirmed by diethyl pyrocarbonate footprinting of the complex between antibodies to Z-DNA and the AC-rich region. Osmium tetroxide and hydroxylamine reveal some distortions of the Z double helix in the (GT/CA)n stretch also. The terminal T residues of the stretch are hyperreactive with osmium tetroxide; the terminal left C residues but not the terminal right C residues are hyperreactive with hydroxylamine. Substitution of a few base pairs in the middle of the (GT/CA)n stretch induces also some distortions of the Z double helix. In the GT/CA-rich sequence, distortion of the Z double helix is also supported by the hyperreactivity of osmium tetroxide with several T and C residues.

The electrochemical oxidation of three proteins: RNAase A, bovine serum albumin (BSA) and Concanavalin A, has been investigated on solid electrodes (gold, platinum, vitreous carbon, carbon paste electrode) using a.c. voltammetry and cyclic voltammetry. All the three proteins are adsorbed on these surfaces, but oxidation peaks on voltammograms are only well developed on carbon paste electrode (CPE), therefore the main experimental results presented here will refer to CPE. Tyrosinyl and tryptophanyl residues can first be oxidized over the range of pH 1 to 7; when oxidation potentials are more positive, other amino acids residues (histidine, cystine…) can also be oxidized. In both cases proteins adsorption is prerequisite. The adsorption mainly takes place at negative potentials (less than potential of zero charge) by the intermediary of electrostatic complex with the surface, this electrostatic adsorption is immediately followed by a rearrangement of the proteins at the electrode surface which leads to the formation of non electrostatic irreversible adsorbed complex.

The a.c. polarogram obtained in the case of chemically methylated Micrococcus lysodeikticus DNA is characterized by the appearance of a supplementary peak. Within the range of pH, studied this peak corresponds to a reduction in the adsorbed state without desorption of the reduced products. This peak is characteristic of the structural modifications of the DNA by methylation at the level of the guanine-cytosine base pairing.

The electrochemical properties of amino acids containing no sulfur atoms have been investigated using stationary or rotating solid electrodes such as Au and vitreous carbon. Among the 20 amino acids present in the proteins only tryptophan and tyrosine are specifically oxidizable at a gold, platinum or carbon electrode. Histidine is oxidizable only at a carbon electrode. In the case of tryptophan and tyrosine the oxidation phenomena are very similar whatever the nature of the electrode. Oxidation can be performed on a smooth surface electrode, and at the level of metal oxide formation it leads to a mixture of monomeric species (oxindolalanyl with tryptophan and hexacyclodienone alanyl with tyrosine) and polymeric species (filming effect: polytryptophan with tryptophan, polyphenylene oxide alanyl with tyrosine). According to experimental features (pH, concentrations, electrode angular velocity, etc.) either the first of the second electrochemical oxidation pathway can be favored.

Molecular modeling is used to study the sequence dependence of conformation and stability within helically regular duplex Z-DNA. The variations of conformation that are found are sufficiently important to be classified as a new type of polymorphism within the Z family. It is also demonstrated that certain sequences can adopt more than one of these polymorphic forms. Comparison with experimental studies of chemical reactivity within a natural DNA fragment, forced into a left-handed conformation, suggests that the results of our modeling may be used to explain the chemical reactivity observed. Comparison of the Z results with similar studies of the B form allow enthalpies of transition to be calculated as a function of base sequence.

By a new potentiostatic double step-sweep method conveniently applicable with common commercial polarographic equipment the adsorption and interfacial behaviour of DNA of different origin and of related biosynthetic polynucleotides has been studied in moderately acid solution (pH 5.6) of different ionic strengths over the whole extended potential range of adsorption up to −1.6 V (SCE) at the HMDE. Furthermore the new method has proved to be an efficient tool to follow the kinetics of the reduction of strongly adsorbed substances to as well strongly adsorbed reduction products. Three potential ranges corresponding to different interfacial situations can be distinguished for the investigated polynucleotides. In the first range between −0.4 and − 1.2 V (SCE) the biopolymer is adsorbed. If the biopolymer has initially a double stranded form progressive irreversible deconformation occurs as well in this potential range furnishing further evidence for the previously developed concept of the sequence of interfacial events. In the second range between −1.2 and −1.6 V biopolymers are adsorbed and (in the double stranded case after prior deconformation) the adenine and cytosine moieties undergo a totally irreversible reduction forming according to the nature of the polynucleotide a more or less completely blocking film of strongly adsorbed reduced biopolymer. In the third range beyond −1.6 V no adsorption and thus reduction occurs. The kinetic parameters k ct and α c n a of the rate determining step of the charge transfer reaction in which the adsorbed biopolymer is reduced have been studied as function of the nature and structure of the polynucleotide, of its base composition and of ionic strength In general the measurements improve significantly the understanding of the complicated behaviour of DNA and related biosynthetic polynucleotides at charged interfaces and confirm in various ways our previous resulis and conclusions.

The electrochemical oxidation of sulfur amino acids has been investigated by ac measurements and cyclic voltammetry at solid electrodes (Au, vitreous carbon). Electrochemical oxidation proceeds first in the adsorbed state and second by diffusion at metal electrodes. Electrochemical oxidation is catalyzed by a labile metal oxide. At vitreous carbon electrode oxidation in the adsorbed state is only present in the case of methionine. XPS studies have shown the presence of Download : Download full-size image as intermediate species. To explain the experimental results a reaction path is proposed.

Form V DNA has been prepared from pBR322 DNA by annealing covalently closed complementary single strands. Specific rabbit antibodies to Z-DNA were shown by radioimmunoassay and electron microscopy to react with form V DNA of pBR322. The bound antibodies were visualized either directly (on synthetic polynucleotides in Z-form), or after reaction with goat anti-rabbit immunoglobulin labeled with ferritin (on form V DNA).

Surface enhanced Raman scattering (SERS) spectroscopy has been used to study the interfacial behaviour of DNA modified by cis-Pt(NH3)2Cl2, (cis-DDP) and [Pt-(dien)Cl]Cl bidentate and monodentate platinum coordination compounds, respectively. Two stereochemical configurations of Pt-DNA complexes can be deduced from the adsorption behaviour of the Pt adducts. The antitumoral inactive [Pt-(dien)Cl]Cl forms an outer complex whereas the antitumoral active cis-DDP favours an inner complex.

Poly d(G-C) d(G-C) in B-form, on one hand, and poly d(G-br5C). poly d(G-br5C) and poly d(G-m5C) . poly d(G-m5C) in Z-form, on another hand, were treated with N-AcO-[3H]AAF and the kinetics of these reactions were followed by radioactivity. Covalent binding of carcinogen to the polymers was evaluated after separation of the reacted polymers from non-reacted carcinogen by thin-layer chromatography. We found that B-form polymer reacts twice faster than the Z-form polymers. Proportions of main adducts in the three polymers are almost the same. Results are discussed in relation to the calculated electrostatic potential minima and steric accessibility at the reactive site (1, 2).

A time-resolved fluorescence study of ethidium bromide (EB) in the presence of poly(dG-dC) and of poly(dG-dC) modified by chlorodiethylenetriamine platinum(II) chloride is presented under solvent conditions in which these polymers adopt the Z-conformation (high ionic strength). It is shown that these polynucleotides can intercalate a very small quantity of EB. The binding parameters have been determined. The fluorescence lifetime of EB is slightly higher when bound to the Z-conformation (≃25 ns) than when bound to the B-conformation (≃23.7 ns). The nature of the salt has been checked. In the presence of 2.5M NaClO4, no transition from the Z-conformation to another conformation is observed when EB is added. On the contrary, in the presence of 4.25M NaCl, EB induces a cooperative transition from the Z-conformation to a conformation characterized by a much higher affinity for EB intercalation. In the case of poly(dG-dC) this last conformation is identical to the one observed at low ionic strength (B-conformation), but in the case of the platinated polymer this conformation is slightly different, as judged by the smaller value of the fluorescence lifetime of the intercalated EB.

Poly(dG-dC) . poly(dG-dC) was modified by chlorodiethylenetriamino platinum (II) chloride, cis-dichlorodiammine platinum (II) and trans-dichlorodiammine platinum (II), respectively. The conformation of these modified poly(dG-dC) . poly(dG-dC) was studied by circular dichroism. In 4 M Na+, the circular dichroism spectra of poly(dG-dC)dien-Pt (0 less than or equal to rb less than or equal to 0.2) are similar (rb is the amount of bound platinum per base). It is concluded that the conformation of these polymers belongs to the Z-family. Dien-Pt complexes stabilize the Z-form. The midpoint of the Z goes to B transition of poly(dG-dC)dien-Pt(0.12) is at 0.2 M NaCl. Moreover another B goes to Z transition is observed at lower salt concentration (midpoint at 6 mM NaCl). In 1 mM phosphate buffer, the stability of Z-poly(dG-dC)dien-Pt(0.12) is greatly affected by the presence of small amounts of EDTA. Poly(dG-dC) . poly(dG-dC) modified by cis-Pt and trans-Pt complexes do not adopt the Z-form even in high salt concentration.

Polysaccharides alone or in the presence of DNA are studied by means of alternating current (ac) polarography. When neutral and basic polysaccharides are use, the polarograms recording the quadratic component of the current display one capacitive peak at −1650 mV (SCE). Acid polysaccharides never show this peak and are desorbed from the electrode at more positive potentials. If dextran is used as a reference, this peak allows the determination of the amount of neutral polysaccharides in solution up to 2 μg/ml. The height of this peak has no relation to the ionic strength or pH of the solution within the investigated range. Likewise, the concentration and molecular weight of DNA enclosed in the solution exert no influence upon the peak height. On the other hand, the presence of polysaccharides causes DNA peaks to decrease considerably, ac polarography can, therefore, be regarded as a quick, convenient, and sensitive method for carrying out the titration of polysaccharides alone or mixed with DNA.

Molecular modeling is used to study the sequence dependence of conformation and stability within helically regular duplex Z-DNA. The variations of conformation that are found are sufficiently important to be classified as a new type of polymorphism within the Z family. It is also demonstrated that certain sequences can adopt more than one of these polymorphic forms. Comparison with experimental studies of chemical reactivity within a natural DNA fragment, forced into a left-handed conformation, suggests that the results of our modeling may be used to explain the chemical reactivity observed. Comparison of the Z results with similar studies of the B form allow enthalpies of transition to be calculated as a function of base sequence.

Two rabbits were immunized with native DNA modified in vitro by cis-dichlorodiammineplatinum(II) (cis-Pt). The interactions between the antiserum and several natural and synthetic nucleic acids were studied primarily by radioimmunoassays. Native DNA's modified by platinum compounds with labile groups in the cis position are recognized by the antiserum. No cross-reaction is found with native DNA's substituted by other platinum compounds [trans-dichlorodiammineplatinum(II), cis-diamminotetrachloroplatinum(IV), and chlorodiethylenetriamminoplatinum(II)] and with natural and synthetic modified polyribonucleotides. cis-Pt-modified oligodeoxyribonucleotides and enzymatically hydrolyzed cis-Pt-modified native DNA do not bind to the antiserum. cis-Pt-modified double-stranded synthetic polydeoxyribonucleotides are either hardly recognized or not recognized at all. The antiserum recognized a modified double-stranded helix in DNA which is not formed in several modified synthetic DNA's and RNA's. At least two different antigenic determinants are present in cis-Pt-modified DNA at a ratio of cis-Pt to bases equal to 0.05. Finally, the antiserum does not react with in vivo cis-Pt-modified DNA.

In this work, antibodies against Z-DNA were used to stain polytene chromosomes of Chironomus thummi thummi . By indirect immunofluorescence we report the first identification of left-handed conformation of DNA in a band region. The Chironomus pattern also contrasts with the general staining observed in Drosophila . In Chironomus the antibodies to Z-DNA bind to one interband region of the chromosome II and two bands regions of the chromosome IV.

Two modified polynucleotides having the Z-DNA conformation (poly [dG-dC] dien Pt and poly [dG-br5dC] . poly [dG-br5dC]) have been used for determination of antibodies to Z-DNA. Such antibodies were found in sera of patients with systemic lupus erythematosus and with Crohn's disease. They were scarcely observed in polyradiculoneuritis and in amyotrophic lateral sclerosis. In Crohn's disease sera, no antibodies to B-DNA were ever found but presence of two different families of antibodies to Z-DNA was demonstrated.