In this study, we report the cloning and nucleotide sequence of PCR-generated 5S rDNA from the Tilapiine cichlid fish, Oreochromis niloticus. Two types of 5S rDNA were detected that differed by insertions and/or deletions and base substitutions within the non-transcribed spacer (NTS). Two 5S rDNA loci were observed by fluorescent in situ hybridization (FISH) in metaphase spreads of tilapia chromosomes. FISH using an 18S rDNA probe and silver nitrate sequential staining of 5S-FISH slides showed three 18S rDNA loci that are not syntenic to the 5S rDNA loci.

DNA is a material that has the potential to be used in nanoelectronic devices as an active component. However, the electronic properties of DNA responsible for its conducting behaviour remain controversial. Here we use a self-consistent quantum molecular dynamics method to study the effect of DNA structure and base sequence on the energy involved when electrons are added or removed from isolated molecules and the transfer of the injected charge along de molecular axis when an electric field is applied. Our results have shown that the DNA molecules of poly(C)-poly(G) on B-form and poly(A)-poly(T) on A-form have the highest energy released when one electron is added or removed from them and their Z-form has the lowest energy released. Besides, when an electric field is applied to a charged DNA molecule along its axis there is electron transfer through the molecule, regardless of the number and sign of the injected charge, the molecular structure and the base sequence. Results from these simulations provide useful information that is hard to obtain from the experiments and needs to be considered for a further modelling aiming to improve charge transport efficiency in nanoelectronic devices based on DNA.; Fundação para a Ciência e a Tecnologia (FCT); Programa Operacional “Ciência ...

Enterococcus faecalis LDR55, a human clinical isolate, is resistant to tetracycline (Tcr), erythromycin (Emr), and high levels (greater than 2,000 micrograms/ml) of spectinomycin (Spr) but not streptomycin. Filter matings between strain LDR55 and E. faecalis OG1-RF produced transconjugants with the following resistance phenotypes: Tcr Emr Spr, Tcr Emr, Tcr Spr, and Tcr only but never Emr or Spr only. The genetic determinant encoding resistance to spectinomycin was cloned in Streptococcus sanguis Challis from pDL55, a 26-kb plasmid harbored by a Tcr Spr transconjugant. Subcloning experiments yielded a 1.1-kb ClaI-NdeI fragment that encoded very high-level Spr in S. sanguis (10 mg/ml) and Escherichia coli (50 mg/ml). Cell extracts of cultures obtained from Spr strains expressed adenylating activity for spectinomycin but not for streptomycin, indicating that Spr was due to an AAD(9) activity. The nucleotide base sequence of the 1.1-kb ClaI-NdeI fragment contained a single 750-base open reading frame. The protein predicted from the open reading frame consisted of 250 amino acids and had a calculated size of approximately 28,000 daltons, similar to the size estimated from maxicell analysis (29,000 daltons). The deduced amino acid sequence of the streptococcal AAD(9) was compared with that of the AAD(9) encoded by staphylococcal transposon Tn554. The two proteins shared approximately 39% amino acid identity...

We report the complete thermodynamic library of all 10 Watson-Crick DNA nearest-neighbor interactions. We obtained the relevant thermodynamic data from calorimetric studies on 19 DNA oligomers and 9 DNA polymers. We show how these thermodynamic data can be used to calculate the stability and predict the temperature-dependent behavior of any DNA duplex structure from knowledge of its base sequence. We illustrate our method of calculation by using the nearest-neighbor data to predict transition enthalpies and free energies for a series of DNA oligomers. These predicted values are in excellent agreement with the corresponding values determined experimentally. This agreement demonstrates that a DNA duplex structure thermodynamically can be considered to be the sum of its nearest-neighbor interactions. Armed with this knowledge and the nearest-neighbor thermodynamic data reported here, scientists now will be able to predict the stability (delta G degree) and the melting behavior (delta H degree) of any DNA duplex structure from inspection of its primary sequence. This capability should prove valuable in numerous applications, such as predicting the stability of a probe-gene complex; selecting optimal conditions for a hybridization experiment; deciding on the minimum length of a probe; predicting the influence of a specific transversion or transition on the stability of an affected DNA region; and predicting the relative stabilities of local domains within a DNA duplex.

Physical maps of the genomes of the two newly discovered primate papovaviruses, SA12 and stump-tailed macaque virus (STMV), were generated by restriction endonuclease analysis. The base sequence homologies among the genomes of SA12, stump-tailed macaque virus, and simian virus 40 (SV40) were studied by heteroduplex analysis. Heteroduplexes between SA12 and SV40 DNAs and stump-tailed macaque virus and SV40 DNAs were constructed and mounted for electron microscopy in various amounts of formamide to achieve a range of effective temperatures. At each effective temperature, the regions of duplex DNA in the heteroduplexes were measured and localized on the SV40 physical and functional maps. By analyzing the data from this study and rom our previous study (N. Newell, C. J. Lai, G. Khoury, and T. J. Kelly Jr., J. Virol. 25:193-201, 1978) on the base sequence homology between the genomes of BK virus and SV40, some general conclusions have been drawn concerning the evolutionary relationships among the genomes of the primate papovaviruses. The extent of homology among the viral genomes does not reflect the phylogenetic relationships of their hosts. At comparable effective temperatures Tm - 33 degrees C), the heteroduplexes between the DNAs of BK virus and SV40 contained the largest amount of duplex (about 90%). The heteroduplexes made between SA12 and SV40 DNAs were slightly less homologous...

This study reports the radiation-chemical yields for DNA single strand breaks (ssb) in crystals of CGCACG:CGTGCG (I) and CACGCG:CGCGTG (II) duplexes, induced by direct ionization using X-rays. The DNA fragmentation products, consisting of 3’- and 5’-phosphate-terminated fragments, were quantified by ion-exchange chromatography using a set of reference compounds. The yields of single strand breaks in I and II are 0.16±0.04 μmol/J and 0.07±.02 μmol/J, respectively. The probability of cleavage at a given site is relatively independent of which of the four bases is at that site. For the very small sample of base sequences studied to date, there is no obvious dependence on base sequence. However, there appears to be an increased frequency of strand breaks at the non-phosphorylated termini of the oligodeoxynucleotides. These results show that direct ionization is efficient at producing single strand breaks in DNA and its action is relatively indiscriminate with respect to base sequence.

It has been generally assumed that product formation in DNA damaged by ionizing radiation is relatively independent of base sequence; i.e., that the yield of a given product depends primarily on the chemical properties of each DNA constituent and not on its base sequence context. We examined this assumption by comparing direct-type end products produced in films of d(CTCTCGAGAG)2 with those produced in films of d(GCACGCGTGC)2. Here we report the product yields in d(CTCTCGAGAG)2 hydrated to Γ = 2.5 and 15, where Γ is the hydration level given in mol H2O/mol nucleotide. Of the 16 products monitored by GC/MS, 7 exhibited statistically significant yields: 8-oxoGua, 8-oxoAde, 5-OHMeUra, 5,6-diHUra, 5,6-diHThy, 5-OHCyt, and 5-OHUra. These yields at Γ = 2.5 are compared with the yields from our previously reported study of d(GCACGCGTGC)2 (after projecting the yields to a CG/AT ratio of 1). The ratio of projected yields, d(CTCTCGAGAG)2 divided by d(GCACGCGTGC)2, are 1.3 ± 0.9, 1.8 ± 0.3, 1.6 ± 0.6, 11.4 ± 4.7, 0.2 ± 0.1, > 28, and 0.8 ± 1.1, respectively. Considering just d(CTCTCGAGAG)2, the ratio of yields at Γ = 2.5 divided by yields at Γ = 15, are 0.7 ± 0.2, 0.5 ± 0.1, 2.3 ± 4.0, 3.4 ± 1.2, 3.5 ± 3.3, 1.2 ± 0.2, and 0.4 ± 0.2...

Nucleotide excision repair (NER) efficiencies of DNA lesions can vary by orders of magnitude, for reasons that remain unclear. An example is the pair of N-(2′-deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF) and N-(2′-deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-C8-AAF) adducts that differ by a single acetyl group. The NER efficiencies in human HeLa cell extracts of these lesions are significantly different when placed at G1, G2 or G3 in the duplex sequence (5′-CTCG1G2CG3CCATC-3′) containing the NarI mutational hot spot. Furthermore, the dG-C8-AAF adduct is a better substrate of NER than dG-C8-AF in all three NarI sequence contexts. The conformations of each of these adducts were investigated by Molecular dynamics (MD) simulation methods. In the base-displaced conformational family, the greater repair susceptibility of dG-C8-AAF in all sequences stems from steric hindrance effects of the acetyl group which significantly diminish the adduct-base stabilizing van der Waals stacking interactions relative to the dG-C8-AF case. Base sequence context effects for each adduct are caused by differences in helix untwisting and minor groove opening that are derived from the differences in stacking patterns. Overall, the greater NER efficiencies are correlated with greater extents of base sequence-dependent local untwisting and minor groove opening together with weaker stacking interactions.

A high copy, tandemly repeated, sequence (Bd49) specific to the B chromosome and located near the centromere in Brachycome dichromosomatica was used to identify lambda genomic clones from DNA of a 3B plant. Only one clone of those analysed was composed entirely of a tandem array of the B-specific repeat unit. In other clones, the Bd49 repeats were linked to, or interspersed with, sequences that are repetitious and distributed elsewhere on the A and B chromosomes. One such repetitious flanking sequence has similarity to retrotransposon sequences and a second is similar to chloroplast DNA sequences. Of the four separate junctions analysed of Bd49-like sequence with flanking sequence, three were associated with the same A/T-rich region in Bd49 and the fourth was close to a 25 bp imperfect dyadic sequence. No novel B-specific sequences were detected within the genomic clones.; Franks, T. ; Houben, A. ; Leach, C. ; Timmis, J.

The search for the carbohydrate-deficient glycoprotein syndrome type I (CDG1) gene has revealed the existence of a family of phosphomannomutase (PMM) genes in humans. Two expressed PMM genes, PMM1 and PMM2 , are located on chromosome bands 22q13 and 16p13, respectively, and a processed pseudogene PMM2 psi is located on chromosome 18p. Mutations in PMM2 are the cause of CDG type IA whereas no disorder has been associated with defects in PMM1 as yet. Here, we describe the genomic organization of these paralogous genes. There is a 65% identity of the coding sequence, and all intron/exon boundaries have been conserved. The processed pseudogene is more closely related to PMM2 . Remarkably, several base substitutions in PMM2 that are associated with disease are also present at the corresponding positions in the pseudogene. Thus, mutations that occur at a slow rate in the active gene in the population have also accumulated in the pseudogene.

We reported the identification of a new family of DNA-binding proteins from our characterization of the dead ringer (dri) gene of Drosophila melanogaster. We show that dri encodes a nuclear protein that contains a sequence-specific DNA-binding domain that bears no similarity to known DNA-binding domains. A number of proteins were found to contain sequences homologous to this domain. Other proteins containing the conserved motif include yeast SWI1, two human retinoblastoma binding proteins, and other mammalian regulatory proteins. A mouse B-cell-specific regulator exhibits 75% identity with DRI over the 137-amino-acid DNA-binding domains of these proteins, indicating a high degree of conservation of this domain. Gel retardation and optimal binding site screens revealed that the in vitro sequence specificity of DRI is strikingly similar to that of many homeodomain proteins, although the sequence and predicted secondary structure do not resemble a homeodomain. The early general expression of dri and the similarity of DRI and homeodomain in vitro DNA-binding specificity compound the problem of understanding the in vivo specificity of action of these proteins. Maternally derived dri product is found throughout the embryo until germ band extension...

To understand the basis of pathogenesis by Legionella longbeachae serogroup 1, the importance of the Mip protein in this species was examined. Amino-terminal analysis of the purified, cloned L. longbeachae serogroup 1 ATCC 33462 Mip protein confirmed that the cloned gene protein was expressed and processed in an Escherichia coli background. DNA sequence analysis of plasmid pIMVS27, containing the entire L. longbeachae serogroup 1 mip gene, revealed a high degree of homology to the mip gene of Legionella pneumophila serogroup 1, 76% homology at the DNA level and 87% identity at the amino acid level. Primer extension analysis determined that the start site of transcription was the same for both species, with some differences observed for the 10 and 35 promoter regions. Primers designed from the mip gene sequence obtained for L. longbeachae serogroup 1 ATCC 33462 were used to amplify the mip genes from L. longbeachae serogroup 2 ATCC 33484 and an Australian clinical isolate of L. longbeachae serogroup 1 A5H5. The mip gene from A5H5 was 100% identical to the type strain sequence. The serogroup 2 strain of L. longbeachae differed by 2 base pairs in third-codon positions. Allelic exchange mutagenesis was used to generate an isogenic mip mutant in ATCC 33462 and strain A5H5. The ATCC mip mutant was unable to infect a strain of Acanthamoebae sp. both in liquid and in a potting mix coculture system...

Background Annotations that describe the function of sequences are enormously important to researchers during laboratory investigations and when making computational inferences. However, there has been little investigation into the data quality of sequence function annotations. Here we have developed a new method of estimating the error rate of curated sequence annotations, and applied this to the Gene Ontology (GO) sequence database (GOSeqLite). This method involved artificially adding errors to sequence annotations at known rates, and used regression to model the impact on the precision of annotations based on BLAST matched sequences. Results We estimated the error rate of curated GO sequence annotations in the GOSeqLite database (March 2006) at between 28% and 30%. Annotations made without use of sequence similarity based methods (non-ISS) had an estimated error rate of between 13% and 18%. Annotations made with the use of sequence similarity methodology (ISS) had an estimated error rate of 49%. Conclusion While the overall error rate is reasonably low, it would be prudent to treat all ISS annotations with caution. Electronic annotators that use ISS annotations as the basis of predictions are likely to have higher false prediction rates...

Three different molecular mass forms of IGF-binding proteins (IGFBPs) were purified from ovine plasma by IGF-I affinity chromatography and reverse-phase HPLC: a 46 kDa doublet and 29 kDa and 24 kDa forms. Amino-terminal sequence analysis confirmed that these proteins were ovine (o)IGFBP-3 (46 kDa) and two molecular size variants of oIGFBP-4. oIGFBP-3 and the 29 kDa form of oIGFBP-4 were shown to be N-glycosylated. Isoelectric points were determined to be at approximately pH 6 for oIGFBP-3 and at pH 7 and pH 7.5 for the 29 and 24 kDa forms of oIGFBP-4 respectively. The two different molecular mass variants of oIGFBP-4 had similar IGF-binding properties. Compared with human IGFBP-3 and oIGFBP-3, the two variants of oIGFBP-4 exhibited lower relative binding to amino-terminally modified IGF-I analogues in a competitive IGF-binding assay. The full protein sequence of oIGFBP-4, as deduced from the cDNA sequence, showed a high degree of identity with rat (90%), human (96%) and bovine (98%) IGFBP-4. The cDNA sequence also showed homology over regions of the 3' non-coding sequence, particularly in comparison with bovine IGFBP-4 (96%). Northern analysis of mRNA for oIGFBP-4 indicated a 2.6 kb major transcript and two minor transcripts of approximately 2.1 and 1.8 kb. oIGFBP-4 mRNA transcripts were detected in adult ewe liver > kidney > lung >> heart and also in several fetal tissues...

A heterozygous deletion of a single base (A4704) from exon 37 of the fibrillin-1 gene was defined in a patient with Marfan syndrome and subsequently in his previously undiagnosed father. The deletion created a cryptic 5' splice site in exon 37 which was utilised in preference to the normal 5' splice site during pre-mRNA processing in skin fibroblasts cultured from the proband. The mutant mRNA showed a 48-bp deletion from the 3' end of exon 37 which was predicted to restore the reading frame in the mutant mRNA and result in the deletion of a 16-amino-acid sequence from a central eight-cysteine repeat motif of the fibrillin-1 molecule. Interestingly, the cryptic 5' splice site in exon 37 and the normal 5' splice site had equally strong consensuses for splice-site selection. The preferential utilisation of the cryptic site is discussed in relation to current theories on the mechanisms involved in pre-mRNA splicing. Analysis by reverse-transcription PCR indicated that, in the patients skin fibroblasts, the steady-state level of the mis-spliced mutant mRNA was close to that from the normal allele. In addition, evidence from immunoblotting and pulse-chase biosynthetic labelling indicated that close to normal amounts of fibrillin-1 were being synthesised and secreted by the cells. However...

By using a shuttle vector system developed in our laboratory, we have carried out studies on the molecular mechanism by which 5-bromodeoxyuridine (BrdUrd) induces mutations in mammalian cells. The target for mutagenesis in these studies was the Escherichia coli gpt gene that was contained within a retroviral shuttle vector and integrated into chromosomal DNA in mouse A9 cells. Shuttle vector-transformed cells expressing the gpt gene were mutagenized with BrdUrd and cells with mutations in the gpt gene were selected. Shuttle vector sequences were recovered from the mutant cells, and the base sequence of the mutant gpt genes was determined. The great majority of the BrdUrd-induced mutations involving single-base changes were found to be G.C----A.T transitions. We have shown that mutagenesis by BrdUrd depends upon perturbation of deoxycytidine metabolism. Thus, the current results suggest that BrdUrd mutagenesis involves mispairing and misincorporation of BrdUrd opposite guanine in DNA, driven by nucleotide pool perturbation caused by BrdUrd and the resulting imbalanced supply of triphosphates available for DNA synthesis. The results also revealed a very high degree of sequence specificity for the BrdUrd mutagenesis. BrdUrd-induced G.C----A.T transitions occurred almost exclusively in sequences with two adjacent guanine residues. Furthermore...

Oligodeoxyribonucleotides covalently linked to an intercalating agent via a polymethylene linker were synthesized. Oligothymidylates attached to an acridine dye (Acr) through the 3'-phosphate group [(Tp)n(CH2) mAcr ] specifically interact with the complementary sequence. The interaction is strongly stabilized by the intercalating agent. By using absorption and fluorescence spectroscopies, it is shown that complex formation between (Tp)n(CH2) mAcr and poly(rA) involves the formation of n A X T base pairs, where n is the number of thymines in the oligonucleotide. The acridine ring intercalates between A X T base pairs. Fluorescence excitation spectra reveal the existence of two environments for the acridine ring, whose relative contributions depend on the linker length (m). The binding of (Tp)4(CH2) mAcr to poly(rA) is analyzed in terms of site binding and cooperative interactions between oligonucleotides along the polynucleotide lattice. Thermodynamic parameters show that the covalent attachment of the acridine ring strongly stabilizes the binding of the oligonucleotide to its complementary sequence. The stabilization depends on the linker length; the compound with m = 5 gives a more stable complex than that with m = 3. These results open the way to the synthesis of a family of molecules exhibiting both high-affinity and high-specificity for a nucleic acid base sequence.

We consider force-induced unzipping transition for a heterogeneous DNA model with a correlated base-sequence. Both finite-range and long-range correlated situations are considered. It is shown that finite-range correlations increase stability of DNA with respect to the external unzipping force. Due to long-range correlations the number of unzipped base-pairs displays two widely different scenarios depending on the details of the base-sequence: either there is no unzipping phase-transition at all, or the transition is realized via a sequence of jumps with magnitude comparable to the size of the system. Both scenarios are different from the behavior of the average number of unzipped base-pairs (non-self-averaging). The results can be relevant for explaining the biological purpose of correlated structures in DNA.; Comment: 22 pages, revtex4, 14 eps figures; reprinted in the June 15, 2004 issue of Virtual Journal of Biological Physics Research

Let BS(m,n) denote the set of base sequences (A;B;C;D), with A and B of length m and C and D of length n. The base sequence conjecture (BSC) asserts that BS(n+1,n) exist (i.e., are non-empty) for all n. This is known to be true for n <= 36 and when n is a Golay number. We show that it is also true for n=37 and n=38. It is worth pointing out that BSC is stronger than the famous Hadamard matrix conjecture. In order to demonstrate the abundance of base sequences, we have previously attached to BS(n+1,n) a graph Gamma_n and computed the Gamma_n for n <= 27. We now extend these computations and determine the Gamma_n for n=28,...,35. We also propose a conjecture describing these graphs in general.; Comment: 19 pages, 10 tables. To appear in Discrete Mathematics.

The possibility that the sliding motion of proteins on DNA is influenced by the base sequence through a base pair reading interaction, is considered. Referring to the case of the T7 RNA-polymerase, we show that the protein should follow a noise-influenced sequence-dependent motion which deviate from the standard random walk usually assumed. The general validity and the implications of the results are discussed.; Comment: 12 pages, 3 figures