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PubMed2004

Somatic DNA recombination yielding circular DNA and deletion of a genomic region in embryonic brain.

In this study, a mouse genomic region is identified that undergoes DNA rearrangement and yields circular DNA in brain during embryogenesis. External region-directed inverse polymerase chain reaction on circular DNA extracted from late embryonic brain tissue repeatedly detected DNA of this region containing recombination joints. Wide-range genomic PCR and digestion-circularization PCR analysis showed this region underwent recombination accompanied with deletion of intervening sequences, including the circularized regions. This region was mapped by fluorescence in situ hybridization to C1 on mouse chromosome 16, where no gene and no physiological DNA rearrangement had been identified. DNA sequence in the region has segmental homology to an orthologous region on human chromosome 3q.13. These observations demonstrated somatic DNA recombination yielding genomic deletions in brain during embryogenesis.

Animals↗
PubMed2003

DNA recombination: holliday junctions dynamics and branch migration.

Holliday junctions are critical intermediates for homologous, site-specific recombination, DNA repair, and replication. A wealth of structural information is available for immobile four-way junctions, but the controversy on the mechanism of branch migration of Holliday junctions remains unsolved. Two models for the mechanism of branch migration were suggested. According to the early model of Alberts-Meselson-Sigal (Sigal, N., and Alberts, B. (1972) J. Mol. Biol. 71, 789-793 and Meselson, M. (1972) J. Mol. Biol. 71, 795-798), exchanging DNA strands around the junction remain parallel during branch migration. Kinetic studies of branch migration (Panyutin, I. G., and Hsieh, P. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 2021-2025) suggest an alternative model in which the junction adopts an extended conformation. We tested these models using a Holliday junction undergoing branch migration and time-lapse atomic force microscopy, an imaging technique capable of imaging DNA dynamics. The single molecule atomic force microscopy experiments performed in the presence and in the absence of divalent cations show that mobile Holliday junctions adopt an unfolded conformation during branch migration that is retained despite a broad range of motion in the arms of the junction. This conformation of the junction remains unchanged until strand separation. The data obtained support the model for branch migration having the extended conformation of the Holliday junction.

Base Sequence↗
PubMedDate not supplied

[Effectiveness of recombinant DNA vaccines against hepatitis B in blood donors in an endemic region of South Brazil].

The objective of this work was to estimate the effectiveness of DNA recombinant anti-HBV vaccines in a retrospective cohort study of 1,012 Brazilian blood donors who completed the vaccination schedule (3 doses + booster of antibody titer < 10IU/L) during the period 1998-2002. The results showed that seroconversion rates were significantly lower among the donors whose antibody titers was measured six months after completing the vaccination scheme and among older donors, particularly those aged over 50. Overall vaccine effectiveness was 88.7%, ranging from 80.6% in the oldest (50 years or over) to 91.4% among the youngest (18-30 years) donors. The booster regimen was effective at reducing the percentage of non-responders. We conclude that vaccine effectiveness was significantly better in younger blood donors and that the anti-HBs testing interval influenced the vaccine effectiveness.

Adolescent↗
PubMedDate not supplied

[Experience with anti-hepatitis-B vaccination using a recombinant DNA vaccine, in students of dentistry at the University of Perugia].

Infection with HBV is still one of the more important professional risk for medical staff. This is particularly true for dentists the more so when they are operating in areas at medium or high prevalence of HBsAg carriers. In such situation patients often ignore their carrier state, when they know it they are not always conscious of the importance of notifying the information to their dentist. The dentists, on the other hand, don't routinely collect a detailed anamnesis of their patients. For these reasons the opportunity of being vaccinated against hepatitis B with a DNA recombinant vaccine was offered to dentistry students of the University of Perugia. All participants were to be negative for hepatitis B markers of infection including HBsAg and anti HBs. All volunteers were to have normal aminotransferase levels, be in good physical condition and give their informed written consent. 48 students turned out to be eligible for vaccination, of them 35 were males, 13 females, mean age 23 years. About two months after the third dose, all the vaccinees had seroconverted and 70.4% of them showed antibody titres between 1,000 greater than or equal to 10,000 UI/l. That indicate that very likely the antibody persistence will be long lasting (at least 5 years). Side effects, light and of short duration, were rarely seen.

Adult↗
PubMed1989

[Reactivity and immunogenicity of Engerix B, the recombinant DNA vaccine against hepatitis B].

A group of 67 health workers with no markers indicating previous hepatitis B infection were vaccinated against hepatitis B with a new DNA recombinant vaccine, Engerix B (commercially manufactured by Smith-Kline-RIT, Belgium). Three injections were given according to the 0-1-6 schedule. One month after the last injection the vaccinees were tested for anti-HBs antibodies by the enzyme-linked assay. Antibody titers equal or less than 10 mIU/ml were found only in three subjects or in 4.5% of them. Titers ranging from 11 to 99 mIU/ml were found in 7 subjects (10.4%), from 100 to 999 mIU/ml in 28 (41.8%) and those equal or more than 1000 mIU/ml in 29 subjects (43.3%). It is inferred that the seroconversion rate is 95.5%. Only one subject did not develop detectable antibodies but three subjects had titers over 10000 mIU/ml. No one developed overt hepatitis B during the trial nor did the high responders experienced inapparent infections. They were tested for anti HBc with negative results. Postvaccinal reactions were mild and almost exclusively local. There were no complications. For its high immunogenicity and acceptable reactogenicity the Engerix B vaccine has a promising future.

Adult↗
PubMed1986

Clinical phase II trial of recombinant DNA interferon (interferon alpha 2b) in patients with metastatic malignant melanoma.

Twenty-four patients with histologically proven metastatic malignant melanoma were included in a Phase II trial of human DNA recombinant interferon (rDNA IFN alpha 2). They were given 10 X 10(6) IU of IFN alpha 2 subcutaneously three times a week until progression of disease or major intolerance developed. Twenty-two patients were evaluable for toxicity and response. General manifestations of intolerance were seen in all the patients. Hematologic toxicity was seen in six patients and therapy had to be interrupted in one patient. Mild liver toxicity was seen in most patients after 2 weeks of treatment. These manifestations disappeared within 2 weeks after treatment was discontinued. A partial response was seen in four cases lasting 2, 4, 4, and 5 months, respectively. There were two complete responses (one skin, one lymph node metastasis) lasting 20 and 6 weeks, respectively. These results indicate a potential role for rDNA IFN alpha 2 in treating patients with metastatic malignant melanoma. However, further trials are required to determine the optimal dose and schedule of administration and modalities of combination.

Adult↗
PubMedDate not supplied

[The serological response to a recombinant DNA vaccine in the native residents of 2 hyperendemic hepatitis B areas in the Peruvian Amazonia].

UNLABELLED: In large areas of the Amazon jungle from Brazil, Colombia, Venezuela and Peru, it has been reported hyperendemic HBV and delta focus. In the Peruvian jungle we found up to 97% of prevalence of HBV, on the northern Jívaro tribes vs the southern tribes, Arawak, (65%). In this paper we studied 226 volunteers from 6 Jívaro and 3 Arawak villages, who accepted to be vaccinated with a DNA recombinant vaccine (ENGERIX B); all of them were previously HBsAg/Ab negatives but 55.5% of them were anti-core (IgG) positive. Our results show 84.9% of seroconversion to anti-HBs, and 73.5% was consider to be immunized (> 10 mIU/ml). Better response was observed in those that were HBcAb negative, coming from a low endemic area. We also observed a good vaccine response although we had to change months the response decreased up to 51.8% of seroimmunity. Higher anti-HBs titles was observed in the southern tribes: MGC of 416.3 mIU/ml. compared with the south: MGC: 182.2 mIU/ml. CONCLUSIONS: It is important to check previously, in hyperendemic areas, HBV markers, specially. It is important to receive the second dose between with the third dose up to 14 month. The immune response overall was good: the ABsAb titles obtained was in average, 290 mIU/ml (10.2-41,00). Apparently the vaccine do not suffer during the difficulties in transportation in the jungle. No side effects we shown during the vaccination program.

Adolescent↗
PubMed1994

Analysis of the role of the NUC1 endo/exonuclease in yeast mitochondrial DNA recombination.

Mitochondrial DNA recombination was reduced in an yeast mutant lacking the NUC1 endo/exonuclease. Between linked markers in either the omega or cob region the frequency of recombination decreased nearly 50% compared to wild-type. Gene conversion frequencies in the var1 gene and in the omega region were also lower in the mutant strain. In particular, the gradient of gene conversion at omega was most affected by the absence of the NUC1 nuclease. In crosses between nuclease-deficient and wild-type strains, gene conversion frequencies at omega were reduced only when the omega+ allele was contributed to the zygote by the nuclease-deficient parent. We propose that the 5' exonuclease activity of the NUC1 nuclease functions during recombination to enlarge heteroduplex tracts following a double-strand break in DNA. In crosses between nuclease-deficient and wild-type strains, the anisotropy in gene conversion frequencies at omega is hypothesized to be due to the slow mixing of parental mitochondrial membranes as they fuse in the zygote.

Base Sequence↗
PubMed1989

Human identification and sex determination of dental pulp, bone marrow and blood stains with a recombinant DNA probe.

Recombinant DNA hybridizing specifically to a 300 nucleotide repeat DNA sequence (BLUR8) of human specificity and to human repeat DNA sequence (pHY10) on the Y chromosome was used for human identification and sex determination of degraded DNA samples of blood stains, dental pulp, and bone marrow. This radioactive technique enabled reliable and sensitive human and sex determination from blood stains that were more than 80 years old. Less than 1 piece of 0.5 cm length thread of blood stain was enough for both tests. DNA from relatively fresh dental pulp and bone marrow was clearly identified. The human identification test, which could recognize up to 0.3 ng DNA correctly, was 3 to 5 times more sensitive than the sex determination test.

Adolescent↗
PubMed1979

Preparation of triple-block DNA polymers using recombinant DNA techniques.

The construction of several recombinant plasmid derivatives containing novel triple-block DNA sequence insertions is described. The protocol for these constructions involves synthesis of a heterogenous mixture of block oligomer duplexes, : formula: (see text), using pancreatic deoxyribonuclease and terminal transferase. The synthetic duplexes were mixed with linearized and dG-tailed vectors and the DNA mixture used to transform E. coli. Triple-block sequences of the type dGidAjdCk.dGkdTjdCi, characterized by DNA sequencing, were inserted into the Bam HI site of pBR322 and next to the lac wild-type and UV5 promoter regions in pRW26 and pRW28. Similarly, sequences were inserted into the Sma I site of pACYC189 and could be excised by cleavage with Sma I since the procudure regenerates the recognition site. The approach provides a technique for the synthesis of a large family of defined sequence triple-block polymers in essentially unlimited amounts. Although these inserts contain sequences which have the potential for forming stable hairpin structures, the recombinant plasmids are stable and appear to replicate normally.

Animals↗
PubMed1991

Detection and identification of Leishmania parasites by in situ hybridization with total and recombinant DNA probes.

In situ hybridization on cultured promastigotes and sandfly smears were performed with nonradioactively labeled total DNA and recombinant DNA probes containing minicircle kinetoplast DNA (kDNA) or nuclear DNA inserts. Total DNA probes lack specificity whereas recombinant nuclear DNA probes work only if they contain repetitive sequences. Minicircle kDNAs of five Leishmania isolates, representative of five Leishmania taxa found in Kenya, were sequenced. Comparison of the sequences showed a 150-bp region with around 80% homology, whereas the rest of the minicircles had about 50% homology. Nevertheless, application of these probes in in situ hybridization assays as tested on Leishmania promastigotes in the vector gave good specificity and hybridization signal. Two types of labeling were tested: incorporation of biotin-labeled dUTP or directly horseradish peroxidase (HRP)-labeled nucleotides. Both techniques provided good sensitivity and signal-to-noise ratio on cultured promastigotes. Hybridization with HRP-labeled kDNA probes gave a superior signal-to-noise ratio if tested on sandfly preparations. This method provided a reliable and fast identification and facilitated the detection of promastigotes in sandflies. The technique presented here may be helpful in rapid identification of Leishmania promastigotes, and thus make epidemiological studies easier and less time consuming.

Animals↗
PubMed2003

The National Institutes of Health system for enhancing the science, safety, and ethics of recombinant DNA research.

Oversight of recombinant DNA research by the National Institutes of Health (NIH) is predicated on ethical and scientific responsibilities that are akin, in many ways, to those that pertain to the oversight of animal research. The NIH system of oversight, which originated more than 25 years ago, is managed by the NIH Office of Biotechnology Activities (OBA), which uses various tools to fulfill its oversight responsibilities. These tools include the NIH Guidelines for Research Involving Recombinant DNA Molecules (NIH Guidelines) and the Recombinant DNA Advisory Committee. The OBA also undertakes special initiatives to promote the analysis and dissemination of information key to our understanding of recombinant DNA, and in particular, human gene transfer research. These initiatives include a new query-capable database, an analytical board of scientific and medical experts, and conferences and symposia on timely scientific, safety, and policy issues. Veterinary scientists can play an important role in the oversight of recombinant DNA research and in enhancing our understanding of the many safety and scientific dimensions of the field. These roles include developing appropriate animal models, reporting key safety data, enhancing institutional biosafety review, and promoting compliance with the NIH Guidelines.

Animal Welfare↗
PubMed2005

Dual roles for DNA polymerase eta in homologous DNA recombination and translesion DNA synthesis.

Chicken B lymphocyte precursors and DT40 cells diversify their immunoglobulin-variable (IgV) genes through homologous recombination (HR)-mediated Ig gene conversion. To identify DNA polymerases that are involved in Ig gene conversion, we created DT40 clones deficient in DNA polymerase eta (poleta), which, in humans, is defective in the variant form of xeroderma pigmentosum (XP-V). Poleta is an error-prone translesion DNA synthesis polymerase that can bypass UV damage-induced lesions and is involved in IgV hypermutation. Like XP-V cells, poleta-disrupted (poleta) clones exhibited hypersensitivity to UV. Remarkably, poleta cells showed a significant decrease in the frequency of both Ig gene conversion and double-strand break-induced HR when compared to wild-type cells, and these defects were reversed by complementation with human poleta. Our findings identify a DNA polymerase that carries out DNA synthesis for physiological HR and provides evidence that a single DNA polymerase can play multiple cellular roles.

Animals↗