Production of transgenic rats by ooplasmic injection of spermatogenic cells exposed to exogenous DNA: a preliminary study

The aim of the present study was to investigate the efficiencies of producing transgenic rats by the ooplasmic injection of sperm heads (intracytoplasmic sperm injection: ICSI) and elongating spermatids (elongating spermatid injection: ELSI) exposed to the EGFP DNA solution. A slightly lower proportion of ICSI oocytes using sperm heads exposed to a concentration of 0.5 microg/ml DNA solution for 1 min developed into offspring (13.3%, 48/361) when compared to that of oocytes injected with nontreated sperm heads (19.4%, 32/165). Eight ICSI offspring were found to be EGFP-carrying transgenic rats (16.7% per offspring; 2.2% per embryo). After a 1-min exposure of the elongating spermatids to 5 microg/ml of DNA solution, 8.8% (45/511) of the ELSI oocytes developed into offspring while 12.7% (22/173) of the ELSI oocytes using nontreated spermatids developed. Six ELSI offspring carried the EGFP DNA (13.3% per offspring; 1.2% per embryo). The conventional pronuclear microinjection of 5 microg/ml of DNA solution resulted in the higher production of offspring (29.7%, 104/350) and the birth of three transgenic rats (2.9% per offspring; 0.9% per embryo). Thus, sperm heads and elongating spermatids were practically useful as the vector of exogenous DNA if the DNA-exposed spermatogenic cells were microinseminated into rat oocytes.     

Introduction of a long synthetic repetitive DNA sequence into cultured tobacco cells

Genome information has been accumulated for many species, and these genes and regulatory sequences are expected to be applied in plants by enhancing or creating new metabolic pathways. We hypothesized that manipulating a long array of repetitive sequences using tethered chromatin modulators would be effective for robust regulation of gene expression in close proximity to the arrays. This approach is based on a human artificial chromosome made of long synthetic repetitive DNA sequences in which we manipulated the chromatin by tethering the modifiers. However, a method for introducing long repetitive DNA sequences into plants has not yet been established. Therefore, we constructed a bacterial artificial chromosome-based binary vector in Escherichia coli cells to generate a construct in which a cassette of marker genes was inserted into 60-kb synthetic human centromeric repetitive DNA. The binary vector was then transferred to Agrobacterium cells and its stable maintenance confirmed. Next, using Agrobacterium-mediated genetic transformation, this construct was successfully introduced into the genome of cultured tobacco BY-2 cells to obtain a large number of stable one-copy strains. ChIP analysis of obtained BY-2 cell lines revealed that the introduced synthetic repetitive DNA has moderate chromatin modification levels with lower heterochromatin (H3K9me2) or euchromatin (H3K4me3) modifications compared to the host centromeric repetitive DNA or an active Tub6 gene, respectively. Such a synthetic DNA sequence with moderate chromatin modification levels is expected to facilitate manipulation of the chromatin structure to either open or closed.     

Introduction of antioxidant-loaded liposomes into endothelial cell surfaces through DNA hybridization

Ischemia–reperfusion damage is a problem in organ transplantation. Reactive oxygen species are produced in cells by blood-mediated reactions at the time of blood reperfusion. In this study, we developed a method to immobilize and internalize antioxidants in endothelial cells, using vitamin E-loaded liposomes. The liposomes loaded with vitamin E and human umbilical vein endothelial cells (HUVECs) were modified with poly(ethylene glycol)–phospholipid conjugates carrying 20-mer of deoxyadenylic acid (oligo(dA)₂₀) and 20-mer of complementary deoxythymidylic acid (oligo(dT)₂₀), respectively. The liposomes were effectively immobilized on HUVECs through DNA hybridization between oligo(dA)₂₀ and oligo(dT)₂₀. The liposomes loaded with vitamin E were gradually internalized into HUVECs. Then, the cells were treated with antimycin A to induce oxidative stress. We found the amount of reactive oxygen species was greatly reduced in HUVECs carrying vitamin E-loaded liposomes.     

Introduction of a plant intron into the luciferase gene ofPhotinus pyralis

We describe a new luciferase reporter gene,luc ^INT, for early detection of luciferase activity inAgrobacterium transformation studies, and present improved techniques for the extraction of luciferase that decrease the time needed to quantitate luciferase activity. Theluc ^INT reporter gene combines the PIV2 intron fromGUS ^INT withluc ^*, the modified luciferase gene.luc ^INTis expressed in plant cells but not inAgrobacterium, allowing earlier detection of gene expression in the presence ofAgrobacterium during transformations in tobacco leaf discs. Stable expression levels ofluc ^INT andluc ^* in tobacco suspension cultures are compared for two different promoters. The nucleotide sequence data for the gene will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number U84006.     

The interaction betweenE. coli spheroplasts and plant protoplasts: A proposed procedure to deliver foreign genes into plant cells

Summary E. coli spheroplasts can be used to deliver DNA vectors into plant protoplasts. The use of fluorescent dyes showed that 25–100% of the protoplast population was associated with 1–9 spheroplasts following incubation with several fusogens. Electron microscopy demonstrated spheroplasts attached to protoplasts via a plasma membrane protrusion after high pH/Ca²⁺ treatment, but PEG-high pH/Ca²⁺ promoted endocytosis of spheroplasts into a plasma membrane bounded vesicle. Ultrastructural profiles showed that fusion between spheroplasts and protoplasts did not occur. Immunofluorescence studies detectedE. coli antigens associated with tobacco protoplasts, and after fusogen treatment the antigens were dispersed within the peripheral cytoplasm. The elimination of residual contaminatingE. coli cells from protoplasts was achieved by lysozyme and antibiotic treatment, thus allowing DNA vector assessment in axenic culture.     

Successful transfection of hepatoma cells after encapsulation of plasmid DNA into negatively charged liposomes

The application of conventional cationic liposomes/DNA complexes in gene transfer was hampered due to their large size, instability, and limited transfection site in vivo. In this report, we described a dialysis-based method and produced small, stable, and negatively charged DNA-containing liposomes composed of low content of cationic lipid and high content of fusogenic lipid. The liposomes were relatively spherical with a condensed core inside, and exhibited small size with narrow particle size distribution. The encapsulation efficiency of the liposomes was 42.53 +/- 2.29%. They were stable and showed enough protective ability to plasmid DNA from degradation after incubation with different amounts of DNase. Twenty-fold higher transfection efficiency for the liposomes was achieved when compared with that of naked plasmid DNA and no toxicities to hepatocellular carcinoma cells were observed. Our results indicate that the negatively charged DNA-containing liposomes can facilitate gene transfer in cultured cells, and may alleviate the drawbacks of the conventional cationic liposomes/DNA complexes for gene delivery in vivo.     

Transformation of plant mitochondria with mitochondrial DNA plasmids via protoplast fusion

Summary Brassica napus cybrid plants which contain novel nucleus-mitochondria-chloroplast combinations have been constructed, via protoplast fusion. Such fusions resulted in mitochondrial DNA plasmids being lost (at a frequency of 12.5%) or, more surprisingly, being transferred from mitochondria of one protoplast population to mitochondria of the other population (at a frequency of 6.1%). Mitochondria containing their new DNA complement became the dominant organelle population in regenerated plants and were faithfully maternally inherited through successive sexnal generations. No concomitant alterations in mitochondrial chromosome organization or nuclear chromosome number occurred. Protoplast fusion can, therefore, cure plant mitochondria of extrachromosomal DNA and, more importantly, be used to transform plant mitochondria with naturally occurring mitochondrial plasmids. The potential for mitochondrial transformation with recombinant vectors is discussed.     

In vitro analysis of a type I DNA topoisomerase activity from cultured tobacco cells

The role of DNA topoisomerases in plant cell metabolism is currently under investigation in our laboratory. Using a purified type I topoisomerase from cultured tobacco, we have carried out a biochemical characterization of enzymatic behavior. The enzyme relaxes negatively supercoiled DNA in the presence of MgCl₂, and to a lesser extent in the presence of KCl. Phosphorylation of the topoisomerase does not influence its activity and it is not stimulated by the presence of histones H1 or H5. The enzyme may act in either a processive or distributive manner depending on reaction conditions. The anti-tumor drug, camptothecin, induces significant breakage by the enzyme on purified DNA molecules unless destabilized by the addition of KCl. The tobacco topoisomerase I can catalyze the formation of stable nucleosomes on circular DNA templates, suggesting a role for the enzyme in chromatin assembly.     

Bacteriophage λ DNA fragments replicate in the Paramecium macronucleus: Absence of active copy number control

We show that bacteriophage λ DNA fragments microinjected into the macronucleus of the ciliated protozoan Paramecium can replicate as unit-length linear molecules. These linear DNA molecules are substrates for the addition of Paramecium telomeres by an endogenous telomerase. The linear DNA pieces can exist at copy numbers much higher than that of typical endogenous macronuclear chromosomes. We show that the copy number of injected DNA many fissions after microinjection reflects that of the original input copy number, suggesting that active control of copy number does not occur. Instead, the results suggest that injected DNA is replicated once per cell division. © 1992 Wiley-Liss, Inc.     

玉米自交系中白芸豆（PhaseolusvulgarisL．）DNA的导入转化及引起性状变异的研究初报

本实验对受体花粉携带法在玉米自交系转育中做了进一步的探讨。提取白芸豆ＤＮＡ,将其与受体花粉混合后授粉,其变异率在９．６×１０－５－５．５６×１０－２之间,若对混合后时间长短做以比较,则变异顺序为２０ｍｉｎ＞０～５ｍｉｎ＞１０～１５ｍｉｎ,若同时考虑到当代结实率、不出苗率等,则花粉与ＤＮＡ混合０～５ｍｉｎ后授粉为宜．变异性状为株高、生育期、株型、育性等微效多基因控制的数量性状,质量性状的变化不明显．     

DNA methylation reprogramming of human cancer cells by expression of a plant 5-methylcytosine DNA glycosylase

Patterns of DNA methylation, an important epigenetic modification involved in gene silencing and development, are disrupted in cancer cells. Understanding the functional significance of aberrant methylation in tumors remains challenging, due in part to the lack of suitable tools to actively modify methylation patterns. DNA demethylation caused by mammalian DNA methyltransferase inhibitors is transient and replication-dependent, whereas that induced by TET enzymes involves oxidized 5mC derivatives that perform poorly understood regulatory functions. Unlike animals, plants possess enzymes that directly excise unoxidized 5mC from DNA, allowing restoration of unmethylated C through base excision repair. Here, we show that expression of Arabidopsis 5mC DNA glycosylase DEMETER (DME) in colon cancer cells demethylates and reactivates hypermethylated silenced loci. Interestingly, DME expression causes genome-wide changes that include both DNA methylation losses and gains, and partially restores the methylation pattern observed in normal tissue. Furthermore, such methylome reprogramming is accompanied by altered cell cycle responses and increased sensibility to anti-tumor drugs, decreased ability to form colonospheres, and tumor growth impairment in vivo. Our study shows that it is possible to reprogram a human cancer DNA methylome by expression of a plant DNA demethylase.     

Selective incorporation of foreign functionality into fibrin gels through a chemically modified DNA aptamer

We found for the first time that a thrombin-binding DNA aptamer (TBA) is selectively entrapped in fibrin gels during the gel growth reaction catalyzed by thrombin. Furthermore, using this phenomenon, we successfully demonstrated multiple incorporation of amphiphilic aliphatic groups into fibrin gels via chemically modified TBA.     

Improvement of the movement and host range properties of a plant virus vector through DNA shuffling

Virus expression vectors based on the tobacco mosaic virus (TMV) genome are powerful tools for foreign gene expression in plants. However, the inclusion of increased genetic load in the form of foreign genes limits the speed of systemic plant invasion and host range of these vectors due to reduced replication and movement efficiencies. To improve these properties of TMV vectors, the gene encoding the 30-kDa movement protein was subjected to mutagenesis and DNA shuffling. A vector that expresses the green fluorescent protein was used to allow simple visual discrimination of mutants with enhanced movement phenotypes. An initial round of mutagenesis produced 53 clones with a faster local movement phenotype. Two subsequent rounds of DNA shuffling produced additional clones that showed further increased rates of cell-to-cell movement and degrees of systemic invasion in restrictive hosts. Surprisingly, sequence analysis of the best performing shuffled genes revealed alterations resulting in coding and silent changes in the movement protein gene. Separation of these coding and silent alterations into distinct gene backgrounds revealed that each contributes to improved movement protein function to differing degrees. The resulting vectors demonstrate that the complex activities of the movement protein genes of viruses can be evolved to have improved movement phenotypes, as evidenced by cell-to-cell and systemic invasion. The experiments produced improved vectors that will be of use both for in planta functional screening and for therapeutic protein production and demonstrated the power of shuffling for plant virus vector improvement.     

Mitochondrial DNA sequence diversity in three ethnic populations from the South-west Iran: a preliminary study

To investigate the genetic structure of human populations in the South-west region of Iran, mitochondrial first hypervariable DNA sequences were obtained from 50 individuals representing three different ethnic groups from Khuzestan Province. Studied groups were Shushtari Persians and Chahar Lang Bakhtiyaries from Indo-European-speaking populations and Bani Torof Arabs from Semitic-speaking linguistic families. Genetic analysis of mtDNA data showed high similarity of Chahar Lang Bakhtiyaries with other Iranian Indo-European-speaking populations while Shushtaries and Bani Torofs had a closer affinity with Semitic-speaking groups rather than to other Iranian populations. The relationship of Chahar Lang Bakhtiyaries and Bani Torof Arabs with their neighbor populations can be explained by linguistic and geographic proximity. Whereas, the greater similarity of Shushtari Persians with West Asian Arabs is probably according to high gene flow between them. This article represents a preliminary study of three major ethnic groups of South-west Iran which investigates the potential genetic substructure of the region.     

Structure and expression of DNA transferred to tobacco via transformation of protoplasts with Ti-plasmid DNA: co-transfer of T-DNA and non T-DNA sequences

Summary The T-DNA structure and organization in tissues obtained via transformation of tobacco protoplasts with Ti-plasmid DNA was found to be completely different from the T-DNA introduced via Agrobacterium tumefaciens. It is often fragmented. Overlapping copies of T-DNA, having various sizes, as well as separated fragments of T-DNA were detected. The border sequences of 23 basepairs (bp), flanking the T-region in the Ti-plasmid as direct repeats are not used as preferred sequences for integration. Similar results were obtained with a T-region clone lacking one of the T_L-borders. This clone, which carried the cytokinin locus and only the right border sequence of T_L and the left border sequence of T_R, still had the capacity to transform protoplasts. Also the Vir-region of the Ti-plasmid is not required for integration of foreign DNA via DNA transformation. This is demonstrated by the results with the T-region clone mentioned and by the transforming capacity of a Ti-plasmid carrying a mutated Vir-region. Nevertheless, in a number of Ti-plasmid DNA transformants Vir-region fragments were found to be stably integrated. Furthermore, it has been established that co-transformation can occur with plant cells. Besides the detection of Ti-plasmid fragments from outside the T-region also DNA sequences originating from two DNA sources, which were both independently present in transformation experiments, have been found in some DNA transformants, e.g. calf thymus DNA, which was used as carrier DNA. No expression of the co-transferred DNA was observed. In total three phenotypical classes of DNA transformants were isolated. Although the T-DNA was often scrambled, polyA⁺ mRNA studies indicated that the different phenotypes studied can be explained by the presence of active T-DNA genes with known functions.