Intracellular Localization of PNA in Human Cells upon its Introduction by Electroporation

Peptide nucleic acid (PNA) is one of the most useful DNA analogs in a wide variety of gene analysis in human cells. In order to exhibit its maximal functions, PNA must be localized to a desired place (e.g., nucleus, cytoplasm and other organelles). Here, we introduced PNAs into HeLa cells by electroporation and examined their localization at various time points. The PNA which binds to the mitochondrial COII gene was initially accumulated in the nucleus, and thereafter mostly transferred to cytoplasm. This time-dependent intracellular localization of PNA is ascribed to the breakdown of the nuclear envelope in the cell division. On the other hand, another PNA that binds to telomere repeat sequence mostly remained in the nucleus, even after the cell division occurred. The retention of this PNA in the nucleus was further enhanced when it was conjugated with Cy3.

(276) Assessing Genetic Diversity of Spinach Germplasm using TRAP Markers

Target region amplified polymorphism (TRAP) markers were used to evaluate genetic variability among 48 accessions of spinach (Spinacia oleracea L.), an economically important leafy vegetable crop in many countries. Thirty-eight accessions collected and preserved by the USDA National Plant Germplasm System (NPGS) and 10 commercial hybrids were used in the current study. For assessing genetic diversity within accessions, DNA samples were prepared from nine to 12 individual seedlings from six germplasm accessions and two hybrids. Relatively high levels of polymorphism was found within accessions based on 61 polymorphic TRAP markers generated with two fixed primers derived from the Arabidopsis-type telomere repeat sequence and two arbitrary primers. For evaluating inter-accession variability, DNA was extracted from a bulk of six to 10 seedlings of each accession. Of the 1092 fragments amplified by 14 primer combinations, 96 (8.8%) were polymorphic and discriminated the 48 accessions from each other. The average pair-wise genetic similarity coefficient (Dice, Nei) was 57.5% with a range from 23.2 to 85.3%. A dendrogram was constructed based on the similarity matrix. It was found that the genetic relationships were not highly correlated with the geographic locations in which the accessions were collected. However, seven commercial hybrids were grouped in three separate clusters, suggesting that the phenotype-based breeding activities have effect on the genetic variability. This study demonstrated that TRAP markers are effective for fingerprinting and evaluating genetic variability of spinach germplasm.

Insertion of a Telomere Repeat Sequence into a Mammalian Gene Causes Chromosome Instability

ABSTRACT Telomere repeat sequences cap the ends of eucaryotic chromosomes and help stabilize them. At interstitial sites, however, they may destabilize chromosomes, as suggested by cytogenetic studies in mammalian cells that correlate interstitial telomere sequence with sites of spontaneous and radiation-induced chromosome rearrangements. In no instance is the length, purity, or orientation of the telomere repeats at these potentially destabilizing interstitial sites known. To determine the effects of a defined interstitial telomere sequence on chromosome instability, as well as other aspects of DNA metabolism, we deposited 800 bp of the functional vertebrate telomere repeat, TTAGGG, in two orientations in the second intron of the adenosine phosphoribosyltransferase (APRT) gene in Chinese hamster ovary cells. In one orientation, the deposited telomere sequence did not interfere with expression of the APRTgene, whereas in the other it reduced mRNA levels slightly. The telomere sequence did not induce chromosome truncation and the seeding of a new telomere at a frequency above the limits of detection. Similarly, the telomere sequence did not alter the rate or distribution of homologous recombination events. The interstitial telomere repeat sequence in both orientations, however, dramatically increased gene rearrangements some 30-fold. Analysis of individual rearrangements confirmed the involvement of the telomere sequence. These studies define the telomere repeat sequence as a destabilizing element in the interior of chromosomes in mammalian cells.