CHROMOSOMAL MAPPING, LINKAGE RELATIONSHIPS, AND EVOLUTIONARY STUDIES OF THE HUMAN ANONYMOUS DNA CLONE D1S1 (IN SITU HYBRIDIZATION, PRIMATES, GENE MAPPING, AUTOSOMAL DOMINANT RETINITIS PIGMENTOSA, GENE DUPLICATION)

D1S1, an anonymous human DNA clone originally called (lamda)Ch4-H3 or (lamda)H3, was the first single copy mapped to a human chromosome (1p36) by in situ hybridization. The chromosomal assignment has been confirmed in other laboratories by repeating the in situ hybridization but not by another method. In the present study, hybridization to a panel of hamster-human somatic cell hybrids revealed copies of D1S1 on both chromosomes 1 and 3. Subcloning D1S1 showed that the D1S1 clone itself is from chromosome 3, and the sequence detected by in situ hybridization is at least two copies of part of the chromosome 3 copy. This finding demonstrates the importance of verifying gene mapping with two methods and questions the accuracy of in situ hybridization mapping.^ Non-human mammals have only one copy of D1S1, and the non-human primate D1S1 map closely resembles the human chromosome 3 copy. Thus, the human chromosome 1 copies appear to be part of a very recent duplication that occurred after the divergence between humans and the other great apes.^ A moderately informative HindIII D1S1 RFLP was mapped to chromosome 3. This marker and 12 protein markers were applied to a linkage study of autosomal dominant retinitis pigmentosa (ADRP). None of the markers proved linkage, but adding the three families examined to previously published data raises the ADRP:Rh lod score to 1.92 at (THETA) = 0.30. ^

CHROMOSOMAL ORIGIN OF DM-DNA

Double minutes (dm) are small chromatin particles of 0.3 microns diameter found only in the metaphase cells of human and murine tumors. Dm are unique cytogenetic structures since their numbers per cell show wide variation. At cell division, dm are retained despite the lack of centromeres. In squash preparations, dm show clustering often in association with chromosomes. Human carcinoma cell line SW613-S18 was found to have large numbers of dm and biological characteristics favorable for mitotic synchronization and chromosome isolation experiments.^ S18 cells were synchronized to mitosis with metabolic and mitotic blocking compounds. Mitotic cells were lysed to release chromosomes and dm from the mitotic spindle and the resulting suspensions were fractionated to enrich for dm. The DNA in enriched fractions was characterized. The reassociation kinetics of dm-DNA driven with placental human DNA was similar to the reassociation curve of labeled placental DNA under similar conditions. In situ hybridization of dm-DNA to tumor and normal metaphase cells showed grain localization over the entire karyotype. Dm-DNA was shown by pulse chase DNA replication experiments to replicate during early and mid S-phase of the cell cycle, but not in late S-phase. In addition, BrdUrd incorporation studies showed that dm-DNA replicates only once during the S-phase. Premature chromosome condensation studies suggest the basis of numerical heterogeneity of dm is nondisjunction, not anomalous or unscheduled DNA replication.^ These data and previous cytochemical banding studies of dm in SW613-S18 indicate that dm-DNA is chromosomal in origin. No evidence of gene amplification was found in the DNA reassociation data. It is likely that dm-DNA represents the pale-staining G-band regions of the human karyotype in this cell line. ^

PCR identification of HIV-1 DNA sequences in brain tissue of patients with AIDS encephalopathy.

We analyzed brain tissue from 39 patients for the presence of proviral HIV-1 sequences, using the polymerase chain reaction (PCR) for the amplification of segments of the viral LTR and gag genes. A novel primer extension procedure allowed the detection of a single HIV-1 copy in 1 micrograms DNA. We detected proviral HIV-1 DNA in 16 of 25 brain samples from AIDS patients. Semiquantitative evaluation of the amplified DNAs indicated considerable variation in viral load. Highest levels of proviral DNA were present in brain samples from six patients with clinical evidence of HIV-associated cognitive/motor complex and the histopathologic correlate of HIV leukoencephalopathy or HIV encephalitis. An additional 11 brain samples contained smaller amounts of proviral DNA. In these patients, clinical data were inconclusive regarding the diagnosis of HIV-1 encephalopathy and histopathologically there was no evidence of HIV-1-induced tissue lesions. In nine of 25 seropositive patients with AIDS (36%), brain samples scored negative or did not contain an unequivocal signal indicating the presence of proviral DNA. HIV-1 sequences were not detected in any of 14 control brain samples from HIV-1 seronegative patients. Our data indicate that HIV-1 is present in the central nervous system of the majority (two thirds) of AIDS patients and that the highest levels of proviral DNA in brain tissue are associated with HIV encephalopathy.

Bipyridyl- and biphenyl-DNA: A recognition motif based on interstrand aromatic stacking

The synthesis and incorporation into oligonucleotides of C-nucleosides containing the two aromatic, non-hydrogen-bonding nucleobase substitutes biphenyl (I) and bipyridyl (Y) are described. Their homo- and hetero-recognition properties in different sequential arrangements were then investigated via UV-melting curve analysis, gel mobility assays, CD- and NMR spectroscopy. An NMR analysis of a dodecamer duplex containing one biphenyl pair in the center, as well as CD data on duplexes with multiple insertions provide further evidence for the zipper-like interstrand stacking motif that we proposed earlier based on molecular modeling. UV-thermal melting experiments with duplexes containing one to up to seven I- or Y base pairs revealed a constant increase in T(m) in the case of I and a constant decrease for Y. Mixed I/Y base pairs lead to stabilities in between the homoseries. Insertion of alternating I/abasic site- or Y/abasic site pairs strongly decreases the thermal stability of duplexes. Asymmetric distribution of I- or Y residues on either strand of the duplex were also investigated in this context. Duplexes with three natural base pairs at both ends and 50 % of I pairs in the center are still readily formed, while duplexes with blunt ended I pairs tend to aggregate unspecifically. Duplexes with one natural overhang at the end of a I-I base pair tract can both aggregate or form ordered duplexes, depending on the nature of the natural bases in the overhang

Pentafluorophenyl phenyl interaction in biphenyl DNA

We prepared and investigated oligonucleotide duplexes of the sequence d(GATGAC(X)(n)GCTAG)d(CTAGC(Y)(n)GTCATC), in which X and Y designate biphenyl- (bph) and pentafluorobiphenyl- ((5F)bph) C-nucleotides, respectively, and n varies from 0-4. These hydrophobic base substitutes are expected to adopt a zipperlike, interstrand stacking motif, in which not only bph/bph or (5F)bph/(5F)bph homo pairs, but also (5F)bph/bph mixed pairs can be formed. By performing UV-melting curve analysis we found that incorporation of a single (5F)bph/(5F)bph pair leads to a duplex that is essentially as stable as the unmodified duplex (n=0), and 2.4 K more stable than the duplex with the nonfluorinated bph/bph pair. The T(m) of the mixed bph/(5F)bph pair was in between the T(m) values of the respective homo pairs. Additional, unnatural aromatic pairs increased the T(m) by +3.0-4.4 K/couple, irrespective of the nature of the aromatic residue. A thermodynamic analysis using isothermal titration calorimetry (ITC) of a series of duplexes with n=3 revealed lower (less negative) duplex formation enthalpies (DeltaH) in the (5F)bph/(5F)bph case than in the bph/bph case, and confirmed the higher thermodynamic stability (DeltaG) of the fluorinated duplex, suggesting it to be of entropic origin. Our data are compatible with a model in which the stacking of (5F)bph versus bph is dominated by dehydration of the aromatic units upon duplex formation. They do not support a model in which van der Waals dispersive forces (induced dipoles) or electrostatic (quadrupole) interactions play a dominant role

Structure/affinity studies in the bicyclo-DNA series: Synthesis and properties of oligonucleotides containing bcen-T and iso-tricyclo-T nucleosides

We present the synthesis of the two novel nucleosides iso-tc-T and bcen-T, belonging to the bicyclo-/tricyclo-DNA molecular platform. In both modifications the torsion around C6’–C7’ within the carbocyclic ring is planarized by either the presence of a C6’–C7’ double bond or a cyclopropane ring. Structural analysis of these two nucleosides by X-ray analysis reveals a clear preference of torsion angle γ for the gauche orientation with the furanose ring in a near perfect 2’-endo conformation. Both modifications were incorporated into oligodeoxynucleotides and their thermal melting behavior with DNA and RNA as complements was assessed. We found that the iso-tc-T modification was significantly more destabilizing in duplex formation compared to the bcen-T modification. In addition, duplexes with complementary RNA were less stable as compared to duplexes with DNA as complement. A structure/affinity analysis, including the already known bc-T and tc-T modifications, does not lead to a clear correlation of the orientation of torsion angle γ with DNA or RNA affinity. There is, however, some correlation between furanose conformation (N- or S-type) and affinity in the sense that a preference for a 3’-endo like conformation is associated with a preference for RNA as complement. As a general rule it appears that Tm data of single modifications with nucleosides of the bicyclo-/tricyclo-DNA platform within deoxyoligonucleotides are not predictive for the stability of fully modified oligonucleotides.

Reactivity of hexanuclear ruthenium metallaprisms towards nucleotides and a DNA decamer

The reactivity of three hexacationic arene ruthenium metallaprisms towards isolated nucleotides and a short DNA strand was investigated using NMR spectroscopy, ESI mass spectrometry, UV/Vis and circular dichroism spectroscopy. The metallaprism built from oxalato-bridging ligands reacts rapidly in the presence of deoxyguanosine monophosphate (dGMP) and deoxyadenosine monophosphate, while the benzoquinonato derivative only reacts with dGMP. On the other hand, the larger metallaprism incorporating naphtoquinonato bridges remains stable in the presence of nucleotides. The reactivity of the three hexacationic metallaprisms with the decameric oligonucleotide d(CGCGATCGCG)2 was also investigated. Analysis of the NMR, MS, UV/Vis and CD data suggests that no adducts are formed between the oligonucleotide and the metallaprisms, but electrostatic interactions, leading to partial unwinding of the double-stranded oligonucleotide, were evidenced

Stability and kinetics of nucleic acid triplexes with chimaeric DNA/RNA third strands

A series of chimaeric DNA/RNA triplex-forming oligonucleotides (TFOs) with identical base-sequence but varying sequential composition of the sugar residues were prepared. The structural, kinetic and thermodynamic properties of triplex formation with their corresponding double-helical DNA target were investigated by spectroscopic methods. Kinetic and thermodynamic data were obtained from analysis of non-equilibrium UV-melting- and annealing curves in the range of pH 5.1 to 6.7 in a 10 mM citrate/phosphate buffer containing 0.1M NaCl and 1 mM EDTA. It was found that already single substitutions of ribo- for deoxyribonucleotides in the TFOs greatly affect stability and kinetics of triplex formation in a strongly sequence dependent manner. Within the sequence context investigated, triplex stability was found to increase when deoxyribonucleotides were present at the 5'-side and ribonucleotides in the center of the TFO. Especially the substitution of thymidines for uridines in the TFO was found to accelerate both, the association and dissociation process, in a strongly position-dependent way. Differential structural information on triplexes and TFO single-strands was obtained from CD-spectroscopy and gel mobility experiments. Only minor changes were observed in the CD spectra of the triplexes at all pH values investigated, and the electrophoretic mobility was nearly identical in all cases, indicating a high degree of structural similarity. In contrast, the single-stranded TFOs showed high structural variability as determined in the same way. The results are discussed in the context of the design of TFOs for therapeutic or biochemical applications.

Bicyclo[3.2.1]amide-DNA: a chiral, non-chiroselective base-pairing system

The design, synthesis and base-pairing properties of bicyclo[3.2.1]amide-(bca)DNA, a novel phosphodiester based DNA analogue, is reported. This analogue consists of a conformationally constrained backbone entity which emulates a B-DNA geometry, to which the nucleobases were attached via an extended, acyclic amide linker. Homobasic adenine-containing bca-decamers form duplexes with complementary oligonucleotides containing the bca-, the DNA the RNA and, surprisingly, also the L-RNA backbone. UV- and CD-spectroscopic investigations revealed the duplexes with D- or L-complement to be of similar stability and enantiomorphic in structure. Bca-oligonucleotides containing all four bases form strictly antiparallel, left-handed complementary duplexes with itself and complementary DNA but not with RNA. Base-mismatch discrimination is comparable to that of DNA while the overall thermal stabilities of bca-oligonucleotide duplexes are inferior relative to that of DNA or RNA. A detailed molecular modeling study of left- and right-handed bca-DNA containing duplexes showed only minor changes in the backbone structure and revealed a structural switch around the base-linker unit to be responsible for the generation of enantiomorphic duplex structures. The obtained data are discussed with respect to the structural and energetic role of the ribofuranose entities in DNA and RNA association

DNA triple-helix formation at pyrimidine-purine inversion sites

A systematic investigation of a series of triplex forming oligonucleotides (TFOs) containing alpha- and beta-thymidine, alpha- and beta-N7-hypoxanthine, and alpha- and beta- N7 and N9 aminopurine nucleosides, designed to bind to T-A inversion sites in DNA target sequences was performed. Data obtained from gel mobility assays indicate that t-A recognition in the antiparallel triple-helical binding motif is possible if the nucleoside alpha N9-aminopurine is used opposite to the inversion site in the TFO.

Distamycin-NA: A DNA Analog with an Aromatic Heterocyclic Polyamide Backbone. Part 1. Synthesis and structural analysis of monomers and dimers containing the nucleobase uracil

The synthesis of the monomeric building block 13 and its constitutional isomer 12 of a new type of DNA analog, distamycin-NA, is presented (Schemes 1 and 2). This building block consists of a uracil base attached to a thiophene core unit via a biaryl-like axis. Next to the biaryl-like axis on the thiophene chromophore, a carboxy and an amino substituent are located allowing for oligomerization via peptide coupling. The proof of constitution and the conformational preferences about the biaryl-like axis were established by means of X-ray analyses of the corresponding nitro derivatives 10 and 11. Thus, the uracil bases are propeller-twisted relative to the thiophene core, and bidentate H-bonds occur between two uracil bases in the crystals. The two amino-acid building blocks 12 and 13 were coupled to give the dimers 15 and 16 using dicyclohexylcarbodiimide (DCC) in THF/LiCl and DMF, respectively. While the dimer 15 showed no atropisomerism on the NMR time scale at room temperature, its isomer 16 occurred as distinct diastereoisomers due to the hindered rotation around its biaryl-like axis. Variable-temperature 1H-NMR experiments allowed to determine a rotational barrier of 19 ± 1 kcal/mol in 16. The experimental data were complemented by AM1 calculations.

About DNA databasing and investigative genetic analysis of externally visible characteristics: A public survey.

During the last decade, DNA profiling and the use of DNA databases have become two of the most employed instruments of police investigations. This very rapid establishment of forensic genetics is yet far from being complete. In the last few years novel types of analyses have been presented to describe phenotypically a possible perpetrator. We conducted the present study among German speaking Swiss residents for two main reasons: firstly, we aimed at getting an impression of the public awareness and acceptance of the Swiss DNA database and the perception of a hypothetical DNA database containing all Swiss residents. Secondly, we wanted to get a broader picture of how people that are not working in the field of forensic genetics think about legal permission to establish phenotypic descriptions of alleged criminals by genetic means. Even though a significant number of study participants did not even know about the existence of the Swiss DNA database, its acceptance appears to be very high. Generally our results suggest that the current forensic use of DNA profiling is considered highly trustworthy. However, the acceptance of a hypothetical universal database would be only as low as about 30% among the 284 respondents to our study, mostly because people are concerned about the security of their genetic data, their privacy or a possible risk of abuse of such a database. Concerning the genetic analysis of externally visible characteristics and biogeographical ancestry, we discover a high degree of acceptance. The acceptance decreases slightly when precise characteristics are presented to the participants in detail. About half of the respondents would be in favor of the moderate use of physical traits analyses only for serious crimes threatening life, health or sexual integrity. The possible risk of discrimination and reinforcement of racism, as discussed by scholars from anthropology, bioethics, law, philosophy and sociology, is mentioned less frequently by the study participants than we would have expected. A national DNA database and the widespread use of DNA analyses for police and justice have an impact on the entire society. Therefore the concerns of lay persons from the respective population should be heard and considered. The aims of this study were to draw a broader picture of the public opinion on DNA databasing and to contribute to the debate about the possible future use of genetics to reveal phenotypic characteristics. Our data might provide an additional perspective for experts involved in regulatory or legislative processes.

Late Quaternary history of North Eurasian Norway spruce ( Picea abies ) and Siberian spruce ( Picea obovata ) inferred from macrofossils, pollen and cytoplasmic DNA variation

Aim We used combined palaeobotanical and genetic data to assess whether Norway spruce (Picea abies) and Siberian spruce (Picea obovata), two major components of the Eurasian boreal forests, occupied separate glacial refugia, and to test previous hypotheses on their distinction, geographical delimitation and introgression. Location The range of Norway spruce in northern Europe and Siberian spruce in northern Asia. Methods Pollen data and recently compiled macrofossil records were summarized for the Last Glacial Maximum (LGM), late glacial and Holocene. Genetic variation was assessed in 50 populations using one maternally (mitochondrial nad1) and one paternally (chloroplast trnT–trnL) inherited marker and analysed using spatial analyses of molecular variance (SAMOVA). Results Macrofossils showed that spruce was present in both northern Europe and Siberia at the LGM. Congruent macrofossil and pollen data from the late glacial suggested widespread expansions of spruce in the East European Plain, West Siberian Plain, southern Siberian mountains and the Baikal region. Colonization was largely completed during the early Holocene, except in the formerly glaciated area of northern Europe. Both DNA markers distinguished two highly differentiated groups that correspond to Norway spruce and Siberian spruce and coincide spatially with separate LGM spruce occurrences. The division of the mtDNA variation was geographically well defined and occurred to the east of the Ural Mountains along the Ob River, whereas the cpDNA variation showed widespread admixture. Genetic diversity of both DNA markers was higher in western than in eastern populations. Main conclusions North Eurasian Norway spruce and Siberian spruce are genetically distinct and occupied separate LGM refugia, Norway spruce on the East European Plain and Siberian spruce in southern Siberia, where they were already widespread during the late glacial. They came into contact in the basin of the Ob River and probably hybridized. The lower genetic diversity in the eastern populations may indicate that Siberian spruce suffered more from past climatic fluctuations than Norway spruce.

Polydnavirus DNA of the braconid wasp Chelonus inanitus is integrated in the wasp's genome and excised only in later pupal and adult stages of the female

Many endoparasitic wasps inject, along with the egg, polydnavirus into their insect hosts, the virus being a prerequisite for successful parasitoid development. The genome of polydnaviruses consists of multiple circular dsDNA molecules of variable size. We show for a 12 kbp segment of the braconid Chelonus inanitus (CiV12) that it is integrated into the wasp genome. This is the first direct demonstration of integration for a bracovirus. PCR data indicated that the integrated form of CiV12 was present in all male and female stages investigated while the excised circular virus DNA only appeared in females after a specific stage in pupal-adult development. The data also indicated that after excision of virus DNA the genomic DNA was rejoined. This has not yet been reported for any polydnavirus. Sequence analyses in the junction regions revealed the presence of an imperfect consensus sequence of 15 nucleotides in CiV12, in each terminus of the integrated virus DNA and in the rejoined genomic DNA. Within these repeats two sequence types (ATA, TAC) were observed in the various virus clones and in the clones encompassing the rejoined genomic DNA; they corresponded to the sequence type in the right and left junction, respectively. To explain this, we propose a model of virus DNA replication in which the genomic DNA is folded to juxtapose the direct repeat of the left with that of the right junction; recombination at specific sites would then yield the two types of virus and rejoined genomic DNA.

Elucidation of the gas-phase structure of a sugar-modified DNA analogue

Antisense oligonucleotides are medical agents for the treatment of genetic diseases that are designed to interact specifically with mRNA. This interaction either induces enzymatic degradation of the targeted RNA or modifies processing pathways, e.g. by inducing alternative splicing of the pre-mRNA. The latter mechanism applies to the treatment of Duchenne muscular dystrophy with a sugar-modified DNA analogue called tricyclo-DNA (tcDNA). In tcDNA the ribose sugar-moiety is extended to a three-membered ring system, which augments the binding affinity and the selectivity of the antisense oligonucleotide for its target. The advent of chemically modified nucleic acids for antisense therapy presents a challenge to diagnostic tools, which must be able to cope with a variety of structural analogues. Mass spectrometry meets this demand for non-enzyme based sequencing methods ideally, because the technique is largely unaffected by structural modifications of the analyte. Sequence coverage of a fully modified tcDNA 15mer can be obtained in a single tandem mass spectrometric experiment. Beyond sequencing experiments, tandem mass spectrometry was applied to elucidate the gas-phase structure and stability of tcDNA:DNA and tcDNA:RNA hybrid duplexes. Most remarkable is the formation of truncated duplexes upon collision-induced dissociation of these structures. Our data suggest that the cleavage site within the duplex is directed by the modified sugar-moiety. Moreover, the formation of truncated duplexes manifests the exceptional stability of the hybrid duplexes in the gas-phase. This stability arises from the modified sugar-moiety, which locks the tcDNA single strand into a conformation that is similar to RNA in A-form duplexes. The conformational particularity of tcDNA in the gas-phase was confirmed by ion mobility-mass spectrometry experiments on tcDNA, DNA, and RNA.