Scattering of repetitive DNA sequences in the albumin and vitellogenin gene loci of Xenopus laevis.

We have analyzed middle repetitive DNA in the albumin and vitellogenin gene families of Xenopus laevis. Mapping specific repetitive DNA sequences derived from introns of the A1 vitellogenin gene reveals that these sequences are scattered within and around the four vitellogenin genes (A1, A2, B1 and B2) and the two albumin genes (74 kd and 68 kd). Three repetitive DNA elements present in the A1 vitellogenin transcriptional unit are also located in introns of the 74 kd albumin gene. This apparently random distribution of middle repetitive DNA in the two gene families suggests that the analyzed sequences are not involved in gene regulation, but rather that they might represent unstable genetic elements. This hypothesis is further supported by the finding that size polymorphism in the A1 vitellogenin gene and in the 74 kd albumin gene is correlated with the presence or absence of repetitive DNA.

Mutations of mitochondrial DNA as potential biomarkers in breast cancer.

BACKGROUND: Alterations of mitochondrial DNA (mtDNA) have been found in cancer patients, therefore informative mtDNA mutations could serve as biomarkers for the disease. MATERIALS AND METHODS: The two hypervariable regions HVR1 and HVR2 in the D-Loop region were sequenced in ten paired tissue and plasma samples from breast cancer patients. RESULTS: MtDNA mutations were found in all patients' samples, suggesting a 100% detection rate. Examining germline mtDNA mutations, a total of 85 mutations in the D-loop region were found; 31 of these mutations were detected in both tissues and matched plasma samples, the other 54 germline mtDNA mutations were found only in the plasma samples. Regarding somatic mtDNA mutations, a total of 42 mutations in the D-loop region were found in breast cancer tissues. CONCLUSION: Somatic mtDNA mutations in the D-loop region were detected in breast cancer tissues but not in the matched plasma samples, suggesting that more sensitive methods will be needed for such detection to be of clinical utility.

Extração e exportação de nutrientes pelo feijoeiro adubado com nitrogênio, em diferentes tempos de implantação do sistema plantio direto

O cultivo do feijoeiro em sistema plantio direto (SPD) tem aumentado de forma marcante no país. Neste contexto, para adubações mais racionais, é fundamental conhecer as exigências nutricionais da cultura quando cultivada em SPD recém-implantado ou consolidado, já que o tempo de implantação do sistema pode alterar a disponibilidade de nutrientes e a resposta das culturas à adubação nitrogenada. Objetivou-se avaliar a extração e exportação de nutrientes pelo feijoeiro em razão da adubação nitrogenada, em solo sob SPD recém-implantado ou consolidado. O experimento foi conduzido por dois anos agrícolas, em um Nitossolo Vermelho distrófico, no município de Botucatu, SP. O delineamento experimental foi em blocos ao acaso, em esquema de parcelas subdivididas, com quatro repetições. As parcelas foram formadas por áreas sob SPD com diferentes tempos de adoção, e as subparcelas constituídas por quatro formas de aplicação do nitrogênio (N) na cultura do feijão (T0: controle, sem aplicação de N; T1: 60 kg ha-1 na pré-semeadura; T2: 60 kg ha-1 aplicado em cobertura no estádio V4; e T3: 60 kg ha-1 na pré-semeadura + 60 kg ha-1 em cobertura). Foram avaliados: matéria seca da parte aérea, concentração e acúmulo de nutrientes na parte aérea, produtividade de grãos e concentrações e exportação de nutrientes nos grãos. O tempo que a área permaneceu sob SPD não influenciou a produtividade, a nutrição e nem mesmo a resposta da cultura do feijão à adubação nitrogenada. A aplicação de N, especialmente em pré-semeadura, proporcionou maiores acúmulos de matéria seca e nutrientes pela cultura do feijão. As concentrações de nutrientes nos grãos foram pouco influenciadas pela adubação nitrogenada. As maiores produtividades de grãos e exportações de nutrientes foram proporcionadas pela aplicação de N em duas épocas (pré-semeadura e em cobertura) ou apenas em cobertura.

Influência do sistema de plantio sobre atributos dendrométricos e fauna edáfica, em área degradada pela extração de argila

O plantio consorciado de eucalipto com leguminosas pode promover a melhoria da qualidade biológica do solo em áreas degradadas e também ser vantajoso para as espécies do consórcio. O objetivo deste trabalho foi avaliar a influência de sistemas de plantios (puros e consorciados) de Acacia mangium (AM), Sesbania virgata (SV) e Eucalyptus camaldulensis (EC), sobre o desenvolvimento das plantas em estudo (variáveis dendrométricas) e especificamente sobre a fauna da serapilheira e dos primeiros 5 cm do solo. Realizou-se um experimento, cujo delineamento utilizado foi o de blocos casualizados com seis tratamentos e três repetições. Os tratamentos utilizados para avaliação dos atributos dendrométricos foram: 100EC (100 % EC) e 100AM (100 % AM); 50EC:50AM (50 % EC + 50 % AM); 50EC:50SV (50 % EC + 50 % SV); e 50AM:50SV (50 % AM + 50 % SV). Para avaliação da fauna do solo foram: 100EC, 100AM, 100SV (100 % SV), 50EC:50SV e 50 AM:50SV. Aos 48 meses após o plantio, foram feitas medições de altura (H) e diâmetro à altura do peito (DAP) das espécies E. camaldulensis e A. mangium e estimativas da área basal (AB) e volume de madeira com casca por indivíduo (VCI). Na serapilheira e no solo (0-5 cm), foram avaliadas a abundância e diversidade da fauna edáfica. O E. camaldulensis quando cultivado em consórcio com as leguminosas apresentou maior DAP, AB e VCI. Em contrapartida, a A. mangium não teve essas variáveis influenciadas quando em consórcio com o E. camaldulensis e com a S. virgata. Plantios de E. camaldulensis e S. virgata em consórcio promoveram maior abundância total de organismos e maiores valores dos índices de diversidade de Shannon e Pielou, principalmente no que se refere ao compartimento serapilheira.

Performance of the 47-Kilodalton Membrane Protein versus DNA Polymerase I Genes for Detection of Treponema pallidum by PCR in Ulcers.

Treponema pallidum PCR (Tp-PCR) is a direct diagnostic method for primary and secondary syphilis, but there is no recommendation regarding the best choice of target gene. In this study, we sequentially tested 272 specimens from patients with sexually transmitted ulcers using Tp-PCR targeting the tpp47 and then polA genes. The two methods showed similar accuracies and an almost-perfect agreement.

Binding of double-strand breaks in DNA by human Rad52 protein.

Double-strand breaks (DSBs) in DNA are caused by ionizing radiation. These chromosomal breaks can kill the cell unless repaired efficiently, and inefficient or inappropriate repair can lead to mutation, gene translocation and cancer. Two proteins that participate in the repair of DSBs are Rad52 and Ku: in lower eukaryotes such as yeast, DSBs are repaired by Rad52-dependent homologous recombination, whereas vertebrates repair DSBs primarily by Ku-dependent non-homologous end-joining. The contribution of homologous recombination to vertebrate DSB repair, however, is important. Biochemical studies indicate that Ku binds to DNA ends and facilitates end-joining. Here we show that human Rad52, like Ku, binds directly to DSBs, protects them from exonuclease attack and facilitates end-to-end interactions. A model for repair is proposed in which either Ku or Rad52 binds the DSB. Ku directs DSBs into the non-homologous end-joining repair pathway, whereas Rad52 initiates repair by homologous recombination. Ku and Rad52, therefore, direct entry into alternative pathways for the repair of DNA breaks.

3D reconstruction and comparison of shapes of DNA minicircles observed by cryo-electron microscopy.

We use cryo-electron microscopy to compare 3D shapes of 158 bp long DNA minicircles that differ only in the sequence within an 18 bp block containing either a TATA box or a catabolite activator protein binding site. We present a sorting algorithm that correlates the reconstructed shapes and groups them into distinct categories. We conclude that the presence of the TATA box sequence, which is believed to be easily bent, does not significantly affect the observed shapes.

The Vi element. A transposon-like repeated DNA sequence interspersed in the vitellogenin locus of Xenopus laevis.

A repeated DNA element in Xenopus laevis is described that is present in about 7500 copies dispersed throughout the genome. It was first identified in the 5' flanking region of one vitellogenin gene and was therefore named the Vi element. Seven copies are present within the vitellogenin gene region, three of them within introns of the genes A1, A2 and B2, and the other four copies in the gene flanking regions. Four of these copies have been sequenced. The Vi element is bounded by a well-conserved 13 base-pair inverted repeat; in addition, it is flanked by a three base-pair direct repeat that appears to be site-specific. The length of these four copies varies from 112 to 469 base-pairs; however, sequence homology between the different copies is very high. Their structural characteristics suggest that length heterogeneity may have arisen by either unequal recombinations, deletions or tandem duplications. Altogether, the characteristics and properties of the Vi element indicate that it might represent a mobile genetic element. One of the four copies sequenced is inserted close (position -535) to the transcription initiation site of the vitellogenin gene B2 in a region otherwise showing considerable homology with the closely related gene B1. Nevertheless, the presence of the Vi element does not seem to influence significantly the estrogen-controlled expression of gene B2. In addition, three alleles of this gene created by length polymorphism in intron 3 and in the Vi element inserted near the transcription initiation site are described.

Protein-protein interactions between adenovirus DNA polymerase and nuclear factor I mediate formation of the DNA replication preinitiation complex.

The in vitro adenovirus (Ad) DNA replication system provides an assay to study the interaction of viral and host replication proteins with the DNA template in the formation of the preinitiation complex. This initiation system requires in addition to the origin DNA sequences 1) Ad DNA polymerase (Pol), 2) Ad preterminal protein (pTP), the covalent acceptor for protein-primed DNA replication, and 3) nuclear factor I (NFI), a host cell protein identical to the CCAAT box-binding transcription factor. The interactions of these proteins were studied by coimmunoprecipitation and Ad origin DNA binding assays. The Ad Pol can bind to origin sequences only in the presence of another protein which can be either pTP or NFI. While NFI alone can bind to its origin recognition sequence, pTP does not specifically recognize DNA unless Ad Pol is present. Thus, protein-protein interactions are necessary for the targetting of either Ad Pol or pTP to the preinitiation complex. DNA footprinting demonstrated that the Ad DNA site recognized by the pTP.Pol complex was within the first 18 bases at the end of the template which constitutes the minimal origin of replication. Mutagenesis studies have defined the Ad Pol interaction site on NFI between amino acids 68-150, which overlaps the DNA binding and replication activation domain of this factor. A putative zinc finger on the Ad Pol has been mutated to a product that fails to bind the Ad origin sequences but still interacts with pTP. These results indicate that both protein-protein and protein-DNA interactions mediate specific recognition of the replication origin by Ad DNA polymerase.

Alkylpurine-DNA-N-glycosylase confers resistance to temozolomide in xenograft models of glioblastoma multiforme and is associated with poor survival in patients.

Glioblastoma multiforme (GBM) is the most common and lethal of all gliomas. The current standard of care includes surgery followed by concomitant radiation and chemotherapy with the DNA alkylating agent temozolomide (TMZ). O⁶-methylguanine-DNA methyltransferase (MGMT) repairs the most cytotoxic of lesions generated by TMZ, O⁶-methylguanine. Methylation of the MGMT promoter in GBM correlates with increased therapeutic sensitivity to alkylating agent therapy. However, several aspects of TMZ sensitivity are not explained by MGMT promoter methylation. Here, we investigated our hypothesis that the base excision repair enzyme alkylpurine-DNA-N-glycosylase (APNG), which repairs the cytotoxic lesions N³-methyladenine and N⁷-methylguanine, may contribute to TMZ resistance. Silencing of APNG in established and primary TMZ-resistant GBM cell lines endogenously expressing MGMT and APNG attenuated repair of TMZ-induced DNA damage and enhanced apoptosis. Reintroducing expression of APNG in TMZ-sensitive GBM lines conferred resistance to TMZ in vitro and in orthotopic xenograft mouse models. In addition, resistance was enhanced with coexpression of MGMT. Evaluation of APNG protein levels in several clinical datasets demonstrated that in patients, high nuclear APNG expression correlated with poorer overall survival compared with patients lacking APNG expression. Loss of APNG expression in a subset of patients was also associated with increased APNG promoter methylation. Collectively, our data demonstrate that APNG contributes to TMZ resistance in GBM and may be useful in the diagnosis and treatment of the disease.

RNA/DNA co-analysis from blood stains--results of a second collaborative EDNAP exercise.

A second collaborative exercise on RNA/DNA co-analysis for body fluid identification and STR profiling was organized by the European DNA Profiling Group (EDNAP). Six human blood stains, two blood dilution series (5-0.001 μl blood) and, optionally, bona fide or mock casework samples of human or non-human origin were analyzed by the participating laboratories using a RNA/DNA co-extraction or solely RNA extraction method. Two novel mRNA multiplexes were used for the identification of blood: a highly sensitive duplex (HBA, HBB) and a moderately sensitive pentaplex (ALAS2, CD3G, ANK1, SPTB and PBGD). The laboratories used different chemistries and instrumentation. All of the 18 participating laboratories were able to successfully isolate and detect mRNA in dried blood stains. Thirteen laboratories simultaneously extracted RNA and DNA from individual stains and were able to utilize mRNA profiling to confirm the presence of blood and to obtain autosomal STR profiles from the blood stain donors. The positive identification of blood and good quality DNA profiles were also obtained from old and compromised casework samples. The method proved to be reproducible and sensitive using different analysis strategies. The results of this collaborative exercise involving a RNA/DNA co-extraction strategy support the potential use of an mRNA based system for the identification of blood in forensic casework that is compatible with current DNA analysis methodology.