Cytosine Research Articles

Is patent foramen ovale closure indicated for migraine?

Is patent foramen ovale closure indicated for migraine?

Effects of tissue specific deaminase/5-fluorocytosine thermotherapy on hepatic metastasis of colonic carcinoma in nude mice.

Objective To investigate the effects of tissue specific cytosine deaminase/5-fluorocytosine (CD/5-FC) thermotherapy on hepatic metastasis of colonic carcinoma in nude mice. Methods Forty-five nude mice were randomly divided into control group, 5-FC group and 5-FC thermotherapy group according to the random number table (15 mice in each group). Mice models of hepatic metastasis of colonic carcinoma were established by portal vein injection of LoVo/CEACD cells. The hepatic metastasis rate and number of metastatic nodules of the 3 groups were compared by ehi-square test and one-way ANOVA. The pathological changes in tumor tissues and apoptotic index of tumor cells were observed. The expression of the CD gene in tumor tissues was detected by fluorescent quantitative RT-PCR and Western blot. Results The number of metastatic nodules and liver metas-tasis rate were 4.6±1.3 and 100.0% in control group, 2.2±1.0 and 60.0% in 5-FC group, 0.5±0.8 and 13.3% in 5-FC thermotherapy group, with statistical difference among the 3 groups (F=25.898, χ2=5.208, 19.548, 5.168, P<0.05). The mean apoptotic indexes of tumor cells of the 3 groups were 4.6%, 9.9% and 17.4%, respectively. Vacuolar degeneration, cell necrosis, cytolysis and apoptotic bodies were mostly observed in the 5-FC thermotherapy group. The expression of CD gene in tumor tissue was detected in all the groups. Conclusion Tissue specific CD/5-FC thermotherapy has inhibitory effects on the hepatic metastasis of LoVo cells transfected with CD gene. Key words: Colonic neoplasms, liver metastasis; Nude mice; Cytosine deaminase; 5-flurocytosine; Gene therapy

Association of the progesterone receptor gene with endometrial cancer risk in a Chinese population

Single nucleotide polymorphisms (SNPs) in the progesterone receptor (PGR) gene have been associated with the risk of endometrial cancer. However, to the authors' knowledge, no study to date has systematically evaluated the role of the PGR gene in endometrial carcinogenesis. Exposure information and DNA samples collected in the Shanghai Endometrial Cancer Study, a population-based case-control study of 1,204 incident cases and 1,212 age- and frequency-matched population controls, were used in this study. Seven tag SNPs were identified for the PGR gene plus the 5-kilobase (kb) flanking regions using the Han Chinese data from the HapMap project with a pairwise correlation coefficient (r(2)) >or= 0.90. These 7 SNPs captured 92% of SNPs in the region with a pairwise r(2) >or= 0.90 or 100% of SNPs with a pairwise r(2) >or= 0.80. Genotyping of polymorphisms was performed by using the Affymetrix MegAllele Targeted Genotyping System. A logistic regression model was used to compute adjusted odds ratios (ORs) and 95% confidence intervals (95% CIs). Of 7 tag SNPs that were assessed, 2 polymorphisms in the 3' flanking region of the PGR gene, reference SNP identification number (rs) 11224561 (rs11224561) and rs471767, were associated with the risk of endometrial cancer. The cytosine/cytosine (CC) genotype of SNP rs11224561 was associated with decreased risk (OR, 0.68; 95% CI, 0.50-0.92) compared with the thymine/thymine (TT) genotype. Carrying the guanine (G) allele of the rs471767 SNP also was associated with decreased risk, although the association was not statistically significant (OR, 0.78, 95%CI, 0.59-1.04 and OR, 0.32, 95%CI, 0.03-3.05 for the adenine [A]G and GG genotypes, respectively, compared with the homozygote AA). The current findings suggested that polymorphisms in the 3' flanking region of the PGR gene may be associated with the risk of endometrial cancer.

Immunolocalization of DNMT1 and DNMT3a in Salivary Gland Neoplasms

Objective: Salivary gland neoplasms pathogenesis has not been well established. DNA methylation occurs when methyl groups are added to cytosine nucleotides in specific areas of the gene by the enzyme DNA methyltransferase (DNMT). This chemical modification can alter gene expression without altering DNA sequence. While DNMT3a is mostly involved in de novo methylation, DNMT1 acts as a maintenance methyltransferase. We aimed to investigate the immunoexpression of DNMT3a and DNMT1 in minor salivary gland neoplasms, comparing it with normal tissue. Material: Forty-four formalin-fixed and paraffin-embedded samples of pleomorphic adenoma, adenoid cystic carcinoma, mucoepidermoid carcinoma and polymorphous low-grade adenocarcinoma were included in the study. The DNMT1 and DNMT3a proteins were identified by using a highly sensitive polymer-based system. Results: Positive nuclear and cytoplasmic labeling for DNMT1 was observed in all samples, including the controls. Positive nuclear labeling for DNMT3a was found only in few neoplasms: 1 pleomorphic adenoma (9.0%), 2 adenoid cystic carcinoma (16.6%) and 1 mucoepidermoid (9.0%) cases. Conclusion: Our results were not able to demonstrate a clear correlation between DNMT1 and DNMT3a immunoexpression and salivary gland neoplasms development.

Structure of the cytosine–cytosine mismatch in the thymidylate synthase mRNA binding site and analysis of its interaction with the aminoglycoside paromomycin

The structure of a cytosine-cytosine (CC) mismatch-containing RNA molecule derived from a hairpin structure in the thymidylate synthase mRNA that binds the aminoglycoside paromomycin with high affinity was determined using nuclear magnetic resonance (NMR) spectroscopy. The cytosines in the mismatch form a noncanonical base pair where both cytosines are uncharged and stack within the stem of the RNA structure. Binding to paromomycin was analyzed using isothermal titration calorimetry (ITC) to demonstrate the necessity of the CC mismatch and to determine the affinity dissociation constant of this RNA to paromomycin to be 0.5 +/- 0.3 microM. The CC mismatch, and the neighboring GC base pairs experienced the highest degree of chemical shift changes in their H6 and H5 resonances indicating that paromomycin binds in the major groove at the CC mismatch site. In comparing the structure of CC mismatch RNA with a fully Watson-Crick GC base paired stem, the CC mismatch is shown to confer a widening of the major groove. This widening, combined with the dynamic nature of the CC mismatch, enables binding of paromomycin to this RNA molecule.

Epigenetic Investigations in Drosophila for Identifying Genes Responsive to Different Stress Conditions

Long-lived organisms tend to be more resistant to various forms of environmental stress. For a while authors reported the absence of methylated cytosine residues in Drosophila melanogaster genome, but recently there have been published several studies which proved that Drosophila genome contain methylated cytosines. With respect to these studies, we conducted an epigenetic screening, trying to test in different Drosophila strains, the response of several genes to unlike stress condition. Four single-copy genes were monitored for their status of methylation: Hsp70, Hsp22, CG3706 and Indy, in three different Drosophila strains, named so that to correspond with the polluted regions from Romania chosen for investigation: Bucharest-Polycolor (B), Seaca (Tr), Braila (Br). For each selected gene we investigated mainly the promoter region. All the results were related to that obtained with the control isogenised strain from the lab, the iso strain. The methods used for investigation were bisulfite sequencing along with molecular cloning and MSP (Methylation Specific PCR). The results demonstrated that the selected genes for this screening are not silenced by methylation, irrespective of the stock selected from different polluted areas. Our results are not so surprisingly if is taken into account that it have been previously shown that in Drosophila only about 0,1-0,6% of all cytosine are methylated, and nowadays are still lots of questions for a specific role of the DNA methylation in flies.

Peeling Single-Stranded DNA from Graphite Surface to Determine Oligonucleotide Binding Energy by Force Spectroscopy

We measured the force required to peel single-stranded DNA molecules from single-crystal graphite using chemical force microscopy. Force traces during retraction of a tip chemically modified with oligonucleotides displayed characteristic plateaus with abrupt force jumps, which we interpreted as a steady state peeling process punctuated by complete detachment of one or more molecules. We were able to differentiate between bases in pyrimidine homopolymers; peeling forces were 85.3 - 4.7 pN for polythymine and 60.8 +/- 5.5 pN for polycytosine, substantially independent of salt concentration and the rate of detachment. We developed a model for peeling a freely jointed chain from the graphite surface and estimated the average binding energy per monomer to be 11.5 +/- 0.6 k(B)T and 8.3 +/- 0.7 k(B)T in the cases of thymine and cytosine nucleotides, respectively. The equilibrium free-energy profile simulated using molecular dynamics had a potential well of 18.9 k(B)T for thymidine, showing that nonelectrostatic interactions dominate the binding. The discrepancy between the experiment and theory indicates that not all bases are adsorbed on the surface or that there is a population of conformations in which they adsorb. Force spectroscopy using oligonucleotides covalently linked to AFM tips provides a flexible and unambiguous means to quantify the strength of interactions between DNA and a number of substrates, potentially including nanomaterials such as carbon nanotubes.

The Antiherpetic Drug Acyclovir Inhibits HIV Replication and Selects the V75I Reverse Transcriptase Multidrug Resistance Mutation

The antiviral drug acyclovir is a guanosine nucleoside analog that potently inhibits herpes simplex virus (HSV) replication. Acyclovir treatment in patients coinfected with HSV and human immunodeficiency virus (HIV) has been observed to alter disease course and decrease HIV viral load, a finding that has been attributed to indirect effects of HSV suppression on HIV replication. Based on this hypothesis, several clinical studies have recently investigated the use of acyclovir for treatment of patients coinfected with HSV and HIV or for prophylaxis against HIV transmission. In this report, we use a single round HIV infectivity assay to show that acyclovir directly inhibits HIV infection with an IC50 of approximately 5 microm. The target of acyclovir in HIV-infected cells is validated as HIV reverse transcriptase (RT) by the emergence of the RT variant V75I under the selective pressure of acyclovir. The V75I mutation is part of the multidrug resistance pathway that enhances viral resistance to many of the best RT inhibitors approved for the treatment of HIV. Biochemical analyses demonstrate that acyclovir triphosphate is a chain terminator substrate for HIV RT and can compete with dGTP for incorporation into DNA. Although acyclovir may prove a useful lead for development of new HIV treatments, the selection of resistant mutants raises a cautionary note to the use of acyclovir monotherapy in patients coinfected with HSV and HIV.

Biophysical characterization of DNA aptamer interactions with vascular endothelial growth factor

The binding of a DNA aptamer (5'-CCGTCTTCCAGACAAGAGTGCAGGG-3') to recombinant human vascular endothelial growth factor (VEGF(165)) was characterized using surface plasmon resonance (SPR), fluorescence anisotropy and isothermal titration calorimetry (ITC). Results from both fluorescence anisotropy and ITC indicated that a single aptamer molecule binds to each VEGF homodimer, unlike other VEGF inhibitors that exhibit 2(ligand):1(VEGF homodimer) stoichiometry. In addition, ITC revealed that the association of the aptamer to VEGF at 20 degrees C is enthalpically driven, with an unfavorable entropy contribution. SPR kinetic studies, with careful control of possible mass transfer effects, demonstrated that the aptamer binds to VEGF with an association rate constant k(on) = 4.79 +/- 0.03 x 10(4) M(-1) s(-1) and a dissociation rate constant k(off) = 5.21 +/- 0.02 x 10(-4) s(-1) at 25 degrees C. Key recognition hot-spots were determined by a combination of aptamer sequence substitutions, truncations, and extensions. Most single-nucleotide substitutions, particularly within an mfold-predicted stem, suppress binding, whereas those within a predicted loop have a minimal effect. The 5'-end of the aptamer plays a key role in VEGF recognition, as a single-nucleotide truncation abolished VEGF binding. Conversely, an 11-fold increase in the association rate (and affinity) is observed with a single cytosine nucleotide extension, due to pairing of the 3'-GGG with 5'-CCC in the extended aptamer. Our approach effectively maps the secondary structural elements in the free aptamer, which present the unpaired interface for high affinity VEGF recognition. These data demonstrate that a directed binding analysis can be used in concert with library screening to characterize and improve aptamer/ligand recognition.

Nucleotide Identification and Orientation Discrimination of DNA Homopolymers Immobilized in a Protein Nanopore

Nanopores have been used as extremely sensitive resistive pulse sensors to detect analytes at the molecular level. There has been interest in using such a scheme to rapidly and inexpensively sequence single molecules of DNA. To establish reference current levels for adenine, cytosine, and thymine nucleotides, we measured the blockage currents following immobilization of single-stranded DNA polyadenine, polycytosine, and polythymine within a protein nanopore in chemical orientations in which either the 3' or the 5' end enters the pore. Immobilization resulted in low-noise measurements, yielding sharply defined current distributions for each base that enabled clear discrimination of the nucleotides in both orientations. In addition, we find that not only is the blockage current for each polyhomonucleotide orientation dependent, but also the changes in orientation affect the blockage currents for each base differently. This dependence can affect the ability to resolve polyadenine and polythymine; with the 5' end entering the pore, the separation between polyadenine and polythymine is double that observed for the 3' orientation. This suggests that, for better resolution, DNA should be threaded through the 5' end first in nanopore DNA sequencing experiments.

Phosphodiester bond rupture in 5′ and 3′ cytosine monophosphate in aqueous environment and the effect of low-energy electron attachment: A Car–Parrinello QM/MM molecular dynamics study

In this study we have explored the effect of low-energy electrons (LEEs) when rupturing the C3′–O3′ and C5′–O5′ bonds in 3′ and 5′ cytosine monophosphate in an aqueous environment. This has been done using a hybrid quantum mechanics/classical mechanics (QM/MM) setup within the framework of Car–Parrinello molecular dynamics (CPMD). Our results are in agreement with experimental findings and indicate that LEEs do not drastically lower the energy barrier for breaking the 3′ or 5′ phosphodiester bonds for single cytosine nucleotides in aqueous environment.

In Vivo Reshaping the Catalytic Site of Nucleoside 2′-Deoxyribosyltransferase for Dideoxy- and Didehydronucleosides via a Single Amino Acid Substitution

Nucleoside 2'-deoxyribosyltransferases catalyze the transfer of 2-deoxyribose between bases and have been widely used as biocatalysts to synthesize a variety of nucleoside analogs. The genes encoding nucleoside 2'-deoxyribosyltransferase (ndt) from Lactobacillus leichmannii and Lactobacillus fermentum underwent random mutagenesis to select variants specialized for the synthesis of 2',3'-dideoxynucleosides. An Escherichia coli strain, auxotrophic for uracil and unable to use 2',3'-dideoxyuridine, cytosine, and 2',3'-dideoxycytidine as a source of uracil was constructed. Randomly mutated lactobacilli ndt libraries from two species, L. leichmannii and L. fermentum, were screened for the production of uracil with 2',3'-dideoxyuridine as a source of uracil. Several mutants suitable for the synthesis of 2',3'-dideoxynucleosides were isolated. The nucleotide sequence of the corresponding genes revealed a single mutation (G --> A transition) leading to the substitution of a small aliphatic amino acid by a nucleophilic one, A15T (L. fermentum) or G9S (L. leichmannii), respectively. We concluded that the "adaptation" of the nucleoside 2'-deoxyribosyltransferase activity to 2,3-dideoxyribosyl transfer requires an additional hydroxyl group on a key amino acid side chain of the protein to overcome the absence of such a group in the corresponding substrate. The evolved proteins also display significantly improved nucleoside 2',3'-didehydro-2',3'-dideoxyribosyltransferase activity.

Fragile X Syndrome: Keys to the Molecular Genetics of Synaptic Plasticity

In the previous column, we briefly discussed fragile X syndrome. Here, we expand on this developmental disorder because it introduces key aspects of synaptic plasticity that are the focus of several future columns. Fragile X syndrome is the most common form of inherited mental retardation. Its clinical symptoms include moderate to severe mental retardation, macroorchidism, and behavioral symptoms that include hyperactivity, gaze aversion, and stereotypies. A fragile site on the X chromosome was discovered in 1969, when magnified views of X chromosomes from affected individuals suggested that a section at the end of the chromosome had separated from the rest of the chromosome. The explanation for the fragile site came with the cloning and characterization of the fragile X mental retardation 1 gene (FMR1) in 1991. Gene sequencing indicates that a portion of the gene is dramatically expanded, with sometimes 1,000 to 2,000 new nucleotides found in affected individuals. The new sequence is of a particular sort: the repeat of the three nucleotides cytosine, guanine, and guanine (called a CGG repeat). The expansion is located immediately adjacent to the regulatory portion of the gene, termed the promoter, and interferes with normal transcription of the gene. The net effect is that little if any message is produced, and little if any protein is translated. The gene is effectively silenced. All humans have a small number of CGG repeats at this site, with between 5 and 50 repeats normally present throughout the population. This short repeated sequence does not interfere with transcription. However, the repeat expands to between 50 and 200 repeats in some individuals. Why this happens is not yet understood, but when it occurs, the repeated element expands in the next generation. Individuals with the initial expansion of 200 repeats are carriers, who were initially thought to be affected only mildly, if at all. However, more recent studies show that carriers often develop fragile X tremor ataxia syndrome characterized by gait ataxia, tremor on the onset of movements, as well as milder cognitive and neurological symptoms. Fragile X syndrome is the second disorder in which an

Waxy gene haplotypes: Associations with pasting properties in an international rice germplasm collection

Associations between RVA pasting properties and three single nucleotide polymorphism (SNP) sites in the Waxy gene intron 1, exon 6, and exon 10 were determined using rice genotypes of diverse geographic origin. A total of four SNP-haplotypes (combination of SNP alleles) were identified that explained high proportions of the variation in RVA pasting properties ( R 2 = 0.574–0.704). A haplotype containing DNA sequence variation in exon 10 (exon 10 cytosine nucleotide) was exclusively found in high-apparent amylose content (AAC) genotypes with a higher RVA viscosity profile compared to the high AAC genotypes with a different haplotype. The exon 10 SNP explained variances in coolpaste and setback (coolpaste–hotpaste) to 0.642 and 0.499, respectively. Across three haplotypes, which contained exon 10 adenine nucleotide, AAC was correlated with peak, hotpaste, breakdown and setback (coolpaste–hotpaste) at r = −0.85, −0.75, −0.79, and 0.49, respectively. Therefore, the exon 10 SNP differentiates high AAC types with a strong RVA profile and thus can be used by molecular breeding programs focused on quality improvement. Additionally, characterizing genotypes by their functional SNPs allowed us to better understand the relationship between the Waxy gene, its chemical product (i.e., AAC) and the functionality created by the product (i.e., pasting properties).

Electronic transport in poly(CG) and poly(CT) DNA segments with diluted basepairing

We present a model for describing electrical conductivity along poly(CG) andpoly(CT) DNA segments with diluted base pairing within a tight-bindingHamiltonian approach. The base pairing is restricted to occurring at a fractionp of the cytosine (C) nucleotides at which a guanine (G) nucleotide is attached. We showthat the Schrödinger equation can be mapped exactly onto that of the one-dimensionalAnderson model with diluted disorder. Using a Green function formalism as well as exactdiagonalization of the full one-dimensional Hamiltonian of finite segments, we compute thedensity of states, the wavefunction of all energy eigenstates and their correspondinglocalization lengths. We show that the effective disorder introduced by the diluted basepairing is much stronger in poly(CG) than in poly(CT) segments, with significantconsequences for the electronic transport properties. The electronic wavepacket remainslocalized in the poly(CT) case, while it acquires a diffusive spread for the poly(CG)-basedsequence.

Specific interactions between silver(i) ions and cytosine–cytosine pairs in DNA duplexes

Very specific binding of the Ag(i) ion unexpectedly stabilized DNA duplexes containing the naturally occurring cytosine-cytosine (C-C) mismatch-base pair; because the C-C pair selectively binds to the Ag(i) ion, we developed a DNA-based Ag(i) sensor that employed an oligodeoxyribonucleotide containing C-C pairs used for Ag(i) binding sites.

DNA Methylation Signatures within the Human Brain

DNA methylation is a heritable modification of genomic DNA central to development, imprinting, transcriptional regulation, chromatin structure, and overall genomic stability. Aberrant DNA methylation of individual genes is a hallmark of cancer and has been shown to play an important role in neurological disorders such as Rett syndrome. Here, we asked whether normal DNA methylation might distinguish individual brain regions. We determined the quantitative DNA methylation levels of 1,505 CpG sites representing 807 genes with diverse functions, including proliferation and differentiation, previously shown to be implicated in human cancer. We initially analyzed 76 brain samples representing cerebral cortex (n=35), cerebellum (n=34), and pons (n=7), along with liver samples (n=3) from 43 individuals. Unsupervised hierarchical analysis showed clustering of 33 of 35 cerebra distinct from the clustering of 33 of 34 cerebella, 7 of 7 pons, and all 3 livers. By use of comparative marker selection and permutation testing, 156 loci representing 118 genes showed statistically significant differences--a >or=17% absolute change in DNA methylation (P<.004)--among brain regions. These results were validated for all six genes tested in a replicate set of 57 samples. Our data suggest that DNA methylation signatures distinguish brain regions and may help account for region-specific functional specialization.

Exponential Decay of GC Content Detected by Strand-Symmetric Substitution Rates Influences the Evolution of Isochore Structure

The distribution of guanine and cytosine nucleotides throughout a genome, or the GC content, is associated with numerous features in mammals; understanding the pattern and evolutionary history of GC content is crucial to our efforts to annotate the genome. The local GC content is decaying toward an equilibrium point, but the causes and rates of this decay, as well as the value of the equilibrium point, remain topics of debate. By comparing the results of 2 methods for estimating local substitution rates, we identify 620 Mb of the human genome in which the rates of the various types of nucleotide substitutions are the same on both strands. These strand-symmetric regions show an exponential decay of local GC content at a pace determined by local substitution rates. DNA segments subjected to higher rates experience disproportionately accelerated decay and are AT rich, whereas segments subjected to lower rates decay more slowly and are GC rich. Although we are unable to draw any conclusions about causal factors, the results support the hypothesis proposed by Khelifi A, Meunier J, Duret L, and Mouchiroud D (2006. GC content evolution of the human and mouse genomes: insights from the study of processed pseudogenes in regions of different recombination rates. J Mol Evol. 62:745-752.) that the isochore structure has been reshaped over time. If rate variation were a determining factor, then the current isochore structure of mammalian genomes could result from the local differences in substitution rates. We predict that under current conditions strand-symmetric portions of the human genome will stabilize at an average GC content of 30% (considerably less than the current 42%), thus confirming that the human genome has not yet reached equilibrium.

Localization of Non-Factor VIII Sequence Involved in the Factor VIII Gene Inversion in Hemophilia A Dogs.

There is a common gene inversion at the telomere of the canine X chromosome in which factor VIII intron 22 DNA recombines with homologous sequence outside the gene. This mutation disrupts factor VIII synthesis and causes severe hemophilia A in dogs, analogous to a common inversion seen in humans (Lozier et al, PNAS 2002 99:12991–6). This mutation appears to be the only spontaneous gene inversion in an animal that replicates a corresponding disease of humans. The coding region for canine factor VIII spans approximately 146 kb and is found at the telomere of the X chromosome between bases 126,063,525 and 125,917,394 in the canine genome project, version 46.2d. Much of the canine factor VIII intron 22 remains unsequenced and the presence of F8A (the putative site of recombination in the factor VIII gene) in this region is inferred from PCR and Southern blot analysis of BAC clones that encompass this region. We have therefore focused on localizing the non-factor VIII site of the recombination and have found its essential elements in sequence located telomeric (upstream) to the canine factor VIII coding start site. We have used the boxer canine genome sequence (version 46.2d) as a template and reference for sequence analysis of BAC clone 291M9 (from normal Doberman pinscher genomic DNA library RPCI-81). Comparison of >100,000 bp of sequence common to the boxer and Doberman in this region showed >99% identity between the two breeds as expected. The BAC clone 291M9 was previously shown by DNA fiber FISH to be the region outside the factor VIII gene that participates in the gene inversion. This clone contains both F8A sequence (the putative site of recombination) as well as the sequence (ch8) that replaces the last four exons of the factor VIII transcript in hemophilia A dogs after the inversion. F8A sequence found in the Doberman BAC clone 291M9 matches sequence starting at nucleotide 126,317,916, in the boxer canine genome project (from RPCI-82 boxer BAC clone XX-145E20). This is outside the factor VIII gene, and approximately 400,000,000 base pairs upstream/telomeric to F8A found within the factor VIII gene. Approximately 20 kb from F8A is ch8, the sequence spliced into the abnormal hemophilia A factor VIII mRNA. The 7.5 kb region where the F8A sequence is found is extremely rich in guanosine and cytosine nucleotides (73% GC content) and has at least six putative CpG islands (as identified by the EMBOSS CpG analysis software (http://www.ebi.ac.uk/emboss/cpgplot/). This is consistent with the finding that F8A gene sequences outside the human factor VIII gene are found in CpG islands. There may be additional copies of the canine F8A sequence in or near this region (as is the case for the human genome), since gaps remain in this region of the known genomic sequence. The high GC content and highly repetitive nature makes sequence analysis and assembly problematic. These properties are consistent with DNA that is susceptible to homologous recombination and gene inversion as is seen with hemophilia in dogs and man. [Display omitted]

Pyrimidine salvage and catabolism in leaves of mangrove species

We examined the metabolic fate of [2- 14C]uridine, [2- 14C]cytidine, [2- 14C]uracil and [2- 14C]dihydrouracil in leaf disks of Bruguiera sexangula in 0 mM or 250 mM NaCl. Uridine was readily converted to uracil nucleotides, and cytidine was converted to cytosine nucleotides and also uracil nucleotides, possibly after conversion to uridine. These nucleotides were utilised for synthesis of RNA and UDP-glucose 3 h after administration of labelled precursors. Degradation of uridine and cytidine was extremely limited. In contrast, salvage of [2- 14C]uracil was low, and up to 45% of uracil was degraded to CO 2. [2- 14C]Dihydrouracil, which is a possible intermediate in the reductive pathway of pyrimidine catabolism, was converted to β-ureidopropionic acid and CO 2. No inhibitory effect of 250 mM NaCl was apparent on pyrimidine metabolism of B. sexangula, although RNA synthesis was reduced slightly. The metabolic fate of [2- 14C]uridine was surveyed in seven other mangrove species, Bruguiera gymnorrhiza, Kandelia candel, Rhizophora stylosa, Sonneratia alba, Avicennia marina, Lumnitzera racemosa and Pemphis acidula. The general profile of [2- 14C]uridine in these mangrove leaves was similar to that in B. sexangula; almost all uridine was salvaged to nucleotides, UDP-glucose and RNA, and catabolism was very low. No or very little inhibitory effect of 250 mM NaCl on uridine salvage was found in leaf disks of mangrove species, except L. racemosa. A stimulatory effect of 250 mM NaCl on uridine salvage was found in P. acidula. In contrast, uptake and metabolism of [2- 14C]uridine, especially a salvage pathway, in the leaves of the non-halophyte plant Populus alba, were drastically impaired by 250 mM NaCl.