BioBrick-based ‘Quick Gene Assembly’ in vitro

Because of the technological limitations of de novo DNA synthesis in (i) making constructs containing tandemly repeated DNA sequence units, (ii) making an unbiased DNA library containing DNA fragments with sequence multiplicity in a specific region of target genes, and (iii) replacing DNA fragments, development of efficient and reliable biochemical gene assembly methods is still anticipated. We succeeded in developing a biological standardized genetic parts that are flanked between a common upstream and downstream nucleotide sequences in an appropriate plasmid DNA vector (BioBrick)-based novel assembly method that can be used to assemble genes composed of 25 tandemly repeated BioBricks in the correct format in vitro. We named our new DNA part assembly system: ‘Quick Gene Assembly (QGA)’. The time required for finishing a sequential fusion of five BioBricks is less than 24 h. We believe that the QGA method could be one of the best methods for ‘gene construction based on engineering principles’ at the present time, and is also a method suitable for automation in the near future.

Phylogenetic relationships between annual and perennial species of Helianthus: evolution of a tandem repeated DNA sequence and cytological hybridization experiments

The amplification and chromosomal localization of tandem repeated DNA sequences from Helianthus annuus (clone HAG004N15) and the physical organization of ribosomal DNA were studied in annual and perennial species of Helianthus . HAG004N15-related sequences, which did not show amplification in other Asteraceae except for Viguiera multiflora , were redundant in all the Helianthus species tested, but their frequency was significantly higher in perennials than in annuals. These sequences were located at the ends and intercalary regions of all chromosome pairs of annual species. A similar pattern was found in the perennials, but a metacentric pair in their complement was not labelled. Ribosomal cistrons were carried on two chromosome pairs in perennials and on three pairs in annuals except for H. annuus, where rDNA loci were on four pairs. No difference was observed between cultivated H. annuus and its wild accessions in the hybridization pattern of the HAG004N15 and ribosomal probes. These findings support the hypothesis that the separation between annual and perennial Helianthus species occurred through interspecific hybridization involving at least one different parent. However, GISH in H. annuus using genomic DNA from the perennial Helianthus giganteus as blocking DNA failed to reveal different genomic assets in annual and perennial species.

48 THE AMOUNT OF TELOMERIC DNA IN LYMPHOCYTES OF DOG CLONES

Controversy regarding the restoration of eroded telomere length of donor cells after the nuclear transfer process has arisen from previous studies of cloned cattle, mice, and pigs. Little is known about telomere lengths in dogs from either natural breeding or somatic cell nuclear transfer (SCNT). In this study, we investigated the amount of telomeric DNA (ATD) in the lymphocytes of growing dog clones and their somatic cell donors. One cloned male Afghan hound dog [Snuppy (Lee et al. 2005 Nature 436, 641)] and 3 cloned female Afghan hound dogs (Jang et al. 2006 Theriogenology; doi:10.1016J.THERIOGENOLOGY.2006.11.006) were obtained from somatic cell nuclear transfer (SCNT) of ear skin fibroblasts. The lymphocytes were recovered from all dog clones: their nuclear donor dogs (male donor dog, female donor dog), and six normal Afghan hound dogs (control, and 10-, 20-, 26-, 49-, 55-, and 58-month-old, respectively). The ATD was analyzed by quantitative fluorescence in situ hybridization (Q-FISH) with a telomeric DNA probe. A telomeric probe containing the TTAGGG repeated DNA sequence was simultaneously amplified and labeled with digoxigenin (DIG) by polymerase chain reaction (PCR) using dog genomic DNA as template, a (GGGTAA)7 primer, and a DIG-labeling kit (Roche, Mannheim, Germany). To analyze the amount of telomeric DNA on the lymphocytes, at least 100 interphase nuclei were examined in each specimen. The image was captured by a digital camera (DP-70, Olympus) and analyzed using MetaMorph (Universal Imaging Co., Downingtown, PA, USA), an image analysis program. Our results indicated that the ATD in normal Afghan hounds gradually decreased with age. Although no difference in ATD was observed between 10- and 26-month-old dogs, the ATD in the 26-month-old dog was significantly higher than that in 49-, 55-, and 58-month-olds (P < 0.05). The mean percentage of telomeric DNA in Snuppy (18-month-old; 2.38%) was significantly higher than that in the nuclear donor dog (49-month-old; 2.12%) but less than that in the age-matched control (20-month-old; 2.72%; P < 0.05). The ATD in 3 female clone dogs (3-, 2-, and 2-month-olds; 3.47, 3.28, and 3.07%) were significantly higher than that in the nuclear donor (26-month-old; 2.65%). In conclusion, the mean percentages of telomeric DNA in dog clones were higher than in nuclear donor dogs, and the ATD of the cloned male dog was different from that in age-matched controls from natural reproduction. The results suggest that the amount of telomeric DNA in dog clones can be restored with the nuclear transfer of cultured donor fibroblasts, but further studies are required as to how telomere reprogramming occurs during the nuclear transfer process.

Characterization of the chromosome complement of Helianthus annuus by in situ hybridization of a tandemly repeated DNA sequence

A tandemly repeated sequence isolated from a clone (HAG004N15) of a nebulized genomic DNA library of sunflower (Helianthus annuus L., 2n = 34) was characterized and used to study the chromosome complement of sunflower. HAG004N15 repeat units (368 bp in length) were found to be highly methylated, and their copy number per haploid (1C) genome was estimated to be 7800. After in situ hybridization of HAG004N15 repeats onto chromosome spreads, signals were observed at the end of both chromosome arms in 4 pairs and at the end of only one arm in 8 other pairs. Signals were also observed at the intercalary (mostly subtelomeric) regions in all pairs, in both arms in 8 pairs, and in only one arm in the other 9 pairs. The short arm of 1 pair was labelled entirely. The chromosomal location of ribosomal DNA was also studied by hybridizing the wheat ribosomal probe pTa71. Four chromosome pairs contained ribosomal cistrons at the end of their shorter arm, but a satellite was seen in only 3 pairs. These hybridization patterns were the same in the 3 sunflower lines studied (HA89, RA20031, and HOR). The chromosomal localization of HAG004N15-related sequences allowed all of the chromosome pairs to be distinguished from each other, in spite of small size and similar morphology.

Description of a tandem repeated DNA sequence of Echinostoma caproni and methods for its detection in snail and plankton samples

Echinostome larval stages in the snail have a great potential as effective competitors for the control of schistosomes and adult worms can cause painful intestinal diseases in humans. Ecology and transmission of the larval stages of trematodes are poorly understood, especially because their identification in field-collected samples by microscopy is difficult. We cloned, sequenced and analysed a 192 bp tandem repreated DNA sequence of Echinostoma caproni (EcSau3A), an often discussed antagonist of Schistosoma mansoni in Biomphalaria snails. PCR primers against this sequence can detect less than 10 fg of E. caproni DNA, 2 miracidia in snails 1 day p.i., 1 metacercaria in 50 mg snail tissue and 1 cercaria in 50 mg plankton with high specificity. Methods described in this study can support the discovery of fundamental ecological principles on distribution, host specificity and epidemiology of E. caproni larvae under field conditions.

Localization of a new highly repeated DNA sequence of Lemur catta (Lemuridae, Strepsirhini)

We have isolated and cloned an 800-bp highly repeated DNA (HRDNA) sequence from Lemur catta (LCA) and described its localization on LCA chromosomes. Lemur catta HRDNA sequences were localized by performing FISH experiments on standard and elongated metaphasic chromosomes using an LCA HRDNA probe (LCASAT). A complex hybridization pattern was detected. A strong pericentromeric hybridization signal was observed on most LCA chromosomes. Chromosomes 7 and 13 were lit in pericentromeric regions, as well as in the interspersed heterochromatin. Chromosomes 1, 3, 4, 17, 19, X, and microchromosomes (20, 25, 26, and 27) showed no signals in the pericentromeric region, but chromosomes 3 and 4 showed a positive hybridization in heterochromatic regions. The 800-bp L. catta HRDNA was species specific. We performed FISH experiments with the LCASAT probe on Eulemur macaco macaco (EMA) andEulemur fulvus fulvus (EFU) metaphases and no positive signal of hybridization was detected. These findings were also confirmed by Southern blot analysis and PCR.Key words: highly repeated DNA, FISH, chromosome rearrangements, heterochromatin, Malagasy lemurs.