Recovery Of Genetic Information Research Articles

Prebiotically Plausible "Patching" of RNA Backbone Cleavage through a 3'-5' Pyrophosphate Linkage.

Achieving multiple cycles of RNA replication within a model protocell would be a critical step toward demonstrating a path from prebiotic chemistry to cellular biology. Any model for early life based on an “RNA world” must account for RNA strand cleavage and hydrolysis, which would degrade primitive genetic information and lead to an accumulation of truncated, phosphate-terminated strands. We show here that cleavage of the phosphodiester backbone is not an end point for RNA replication. Instead, 3′-phosphate-terminated RNA strands can participate in template-directed copying reactions with activated ribonucleotide monomers. These reactions form a pyrophosphate linkage, the stability of which we have characterized in the context of RNA copying chemistry. The presence of free magnesium cations results in cleavage of the pyrophosphate bond within minutes. However, we found that the pyrophosphate bond is relatively stable within an RNA duplex and in the presence of chelated magnesium. We show that, under these conditions, pyrophosphate-linked RNA can act as a template for the polymerization of ribonucleotides into canonical 3′–5′ phosphodiester-linked RNA. We suggest that primer extension of 3′-phosphate-terminated RNA followed by template-directed copying represents a plausible nonenzymatic pathway for the salvage and recovery of genetic information following strand cleavage.

HIV-1 RNA genome dimerizes on the plasma membrane in the presence of Gag protein

Retroviruses package a dimeric genome comprising two copies of the viral RNA. Each RNA contains all of the genetic information for viral replication. Packaging a dimeric genome allows the recovery of genetic information from damaged RNA genomes during DNA synthesis and promotes frequent recombination to increase diversity in the viral population. Therefore, the strategy of packaging dimeric RNA affects viral replication and viral evolution. Although its biological importance is appreciated, very little is known about the genome dimerization process. HIV-1 RNA genomes dimerize before packaging into virions, and RNA interacts with the viral structural protein Gag in the cytoplasm. Thus, it is often hypothesized that RNAs dimerize in the cytoplasm and the RNA-Gag complex is transported to the plasma membrane for virus assembly. In this report, we tagged HIV-1 RNAs with fluorescent proteins, via interactions of RNA-binding proteins and motifs in the RNA genomes, and studied their behavior at the plasma membrane by using total internal reflection fluorescence microscopy. We showed that HIV-1 RNAs dimerize not in the cytoplasm but on the plasma membrane. Dynamic interactions occur among HIV-1 RNAs, and stabilization of the RNA dimer requires Gag protein. Dimerization often occurs at an early stage of the virus assembly process. Furthermore, the dimerization process is probably mediated by the interactions of two RNA-Gag complexes, rather than two RNAs. These findings advance the current understanding of HIV-1 assembly and reveal important insights into viral replication mechanisms.

Challenging DNA samples solved with MiniSTR analysis. Brief overview

The recovery of genetic information from highly degraded or minute amount of biological samples needs special approaches together with conventional STR multiplex assays. In this respect, the mini-STR technology is considered to be remarkable for its capacity to decrease the rate of failures and inconclusive results in forensic casework. By using miniSTRs, the PCR efficiency is significantly improved as shorter PCR amplicons are generated with STR primers located closer to the repeat regions. In this article we aimed to present several practical considerations regarding the miniSTR genotyping for challenging forensic samples. Looking for a robust protocol to re-investigate some of our cases with inconclusive or no typing results we studied the efficiency of a miniSTR approach. We report by comparison our results obtained with a commercial miniSTR multiplex assay on samples previously tested with two different 16 STR commercial kits. Our conclusion is definitely in favor of including in the routine protocol the miniSTR assay as it was able to improve the results, providing better or even complete DNA profiles for the difficult samples where the conventional STR kits failed.

Biomolecules in fossil remains: Multidisciplinary approach to endurance

Svante Pääbo, a leading pioneer in the study of ancient DNA, eloquently described the recovery of genetic information from the fossil record as a 21st Century form of genetic time travel1. The advent of PCR made possible the amplification of small amounts of DNA from fossil samples and allowed the direct study of phylogenetics from extinct organisms. Prior to this development, phylogenetic relationships determined by genetic variation relied mostly upon sequences from living organisms. The concept of time travel, via the analysis of ancient biomolecules, can be broadened to encompass numerous types of biomolecular information recovered from ancient bones. For example, palaeodiets and palaeoclimates can be reconstructed from stable isotopes of bone collagen, and estimations of age are obtained from amino acid racemization rates.

Mitochondrial DNA from Myotragus balearicus, an extinct bovid from the Balearic Islands.

DNA was extracted from teeth and bones of Myotragus balearicus, a bovid that evolved in isolation on the Balearic Islands (Western Mediterranean) from the end of the Miocene, becoming extinct 4,000 years BP, after the arrival of humans in the islands. The numerous morphological apomorphies of Myotragus, most strikingly its dwarfism, frontal eyes, and ever-growing incisors, obscure its phylogenetic relationships with extant bovids. Therefore, the recovery of genetic information from Myotragus is of significant interest to help clarify the taxonomic position of this species. In this study we amplified and sequenced a 95 bp (base pair) fragment of the mtDNA cytochrome b gene from 6,000-year-old specimens of Myotragus. Several experimental controls, such as amino acid analysis, independent reproduction in two different laboratories, and cloning of the PCR product, support the authenticity of the ancient DNA sequence recovered. Phylogenetic comparison with orthologous sequences from supposedly related extant genera (serow, goral, mountain goat, chamois, takin, sheep, goat, Himalayan tahr, arctic musk ox, barbary sheep, blue sheep, and saiga) from the Caprinae subfamily suggests that Myotragus is related to some of these species. However, the real phylogenetic position of Myotragus is difficult to assess, due to the lack of resolution of the present molecular study, which can be partially attributed to the short length of the genetic fragment recovered.