Tomato protoplast DNA transformation: physical linkage and recombination of exogenous DNA sequences

We analysed the fate, expression and germ line transmission of exogenous DNA which was microinjected into fertilized eggs of Xenopus laevis. DNA was injected into fertilized eggs within 1 h following fertilization. The injected DNA was dispersed around the site of injection and became localized to cleavage nuclei by stage 6. Injected DNA persisted in the tissues of 6- to 8-month-old frogs and exhibited a mosaic pattern of distribution with regard to the presence or absence and copy number between different tissues. We detected the exogenous DNA sequences in 60% of injected frogs. Restriction digestion analysis of this DNA suggested that it is not rearranged and was organized as head-to-tail multimers. The copy number varied from 2 to 30 copies/cell in various tissues of the same frog. Plasmid pSV2CAT which contains the prokaryotic gene coding for chloramphenicol acetyl transferase (CAT) enzyme linked to the SV40 early gene promoter was expressed in 50% of the animals containing the gene. The pattern of expression, however, varied between different animals and could not be correlated with copy number. We also showed that the exogenous DNA sequences were transmitted through the male germ line and that each offspring contained the gene integrated into a different region of the genome.

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Direct introduction of cloned DNA into the sea urchin zygote nucleus, and fate of injected DNA

Development ◽

10.1242/dev.102.2.287 ◽

1988 ◽

Vol 102 (2) ◽

pp. 287-299 ◽

Cited By ~ 2

Author(s):

R.R. Franks ◽

B.R. Hough-Evans ◽

R.J. Britten ◽

E.H. Davidson

Keyword(s):

Dna Sequences ◽

Larval Growth ◽

Exogenous Dna ◽

Zygote Nucleus ◽

Cell Lineages ◽

Larval Stages ◽

Injection Process ◽

End To End ◽

Nuclear Injection ◽

Cytoplasmic Injection

A method is described for microinjection of cloned DNA into the zygote nucleus of Lytechinus variegatus. Eggs of this species are unusually transparent, facilitating visual monitoring of the injection process. The initial fate of injected DNA fragments appears similar to that observed earlier for exogenous DNA injected into unfertilized egg cytoplasm. Thus after end-to-end ligation, it is replicated after a lag of several hours to an extent indicating that it probably participates in most of the later rounds of DNA synthesis undergone by the host cell genomes during cleavage. The different consequences of nuclear versus cytoplasmic injection are evident at advanced larval stages. Larvae descendant from eggs in which exogenous DNA was injected into the nuclei are four times more likely (32% versus 8%) to retain this DNA in cell lineages that replicate very extensively during larval growth, i.e. the lineages contributing to the imaginal rudiment, and thus to display greatly enhanced contents of the exogenous DNA. Similarly, 36% of postmetamorphic juveniles from a nuclear injection sample retained the exogenous DNA sequences, compared to 12% of juveniles from a cytoplasmic injection sample. However, the number of copies of the exogenous DNA sequences retained per average genome in postmetamorphic juveniles was usually less than 0.1 (range 0.05-50), and genome blot hybridizations indicate that these sequences are organized as integrated, randomly oriented, end-to-end molecular concatenates. It follows that only a small fraction of the cells of the average juvenile usually retains the exogenous sequences. Thus, even when introduced by nuclear microinjection, the stable incorporation of exogenous DNA in the embryo occurs in a mosaic fashion, although in many recipients the DNA enters a wider range of cell lineages than is typical after cytoplasmic injection. Nuclear injection would probably be the route of choice for studies of exogenous DNA function in the postembryonic larval rudiment.

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Tracking the Distribution and Burst of Nuclear Mitochondrial DNA Sequences (NUMTs) in Fig Wasp Genomes

Insects ◽

10.3390/insects11100680 ◽

2020 ◽

Vol 11 (10) ◽

pp. 680

Author(s):

Jian-Xia Wang ◽

Jing Liu ◽

Yun-Heng Miao ◽

Da-Wei Huang ◽

Jin-Hua Xiao

Keyword(s):

Mitochondrial Dna ◽

Dna Sequences ◽

Theoretical Basis ◽

Nuclear Genome ◽

Distribution Patterns ◽

Good Evidence ◽

Fig Wasp ◽

Fig Wasps ◽

Exogenous Dna ◽

Specific Distribution

Mitochondrial DNA sequences can be transferred into the nuclear genome, giving rise to nuclear mitochondrial DNA sequences (NUMTs). NUMTs have been described in numerous eukaryotes. However, the studies on the distribution of NUMTs and its influencing factors are still inadequate and even controversial. Previous studies have suggested that Hymenoptera may be a group rich in NUMTs, in which we selected 11 species of fig wasps (Chalcidoidea, Hymenoptera) to analyze the distribution and evolution of NUMTs at the genomic level. The results showed that the contents of NUMTs varied greatly in these species, and bursts of NUMTs existed in some species or lineages. Further detailed analyses showed that the large number of NUMTs might be related to the large genomes; NUMTs tended to be inserted into unstable regions of the genomes; and the inserted NUMTs might also be affected by transposable elements (TEs) in the neighbors, leading to fragmentations and duplications, followed by bursts of NUMTs. In summary, our results suggest that a variety of genomic environmental factors can determine the insertion and post-insertion fate of NUMTs, resulting in their species- or lineage-specific distribution patterns, and that studying the evolution of NUMTs can provide good evidence and theoretical basis for exploring the dynamics of exogenous DNA entering into the nuclear genome.

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399 SPERMATOZOA-MEDIATED GENE TRANSFER IS INFLUENCED BY SIZE, STRUCTURE, AND CONCENTRATION OF EXOGENOUS DNA

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab399 ◽

2007 ◽

Vol 19 (1) ◽

pp. 315

Author(s):

W. B. Feitosa ◽

M. P. Milazzotto ◽

E. A. L. Martins ◽

A. M. Rocha ◽

J. L. Avanzo ◽

...

Keyword(s):

Dna Sequences ◽

Gel Electrophoresis ◽

Sperm Cell ◽

Reference Data ◽

Size Structure ◽

Specific Protein ◽

Sperm Cells ◽

Exogenous Dna ◽

Mammalian Sperm ◽

Cell Association

Mammalian sperm cells have a spontaneous ability to take up exogenous DNA in a process regulated by specific protein. However, little is known about the effect of exogenous DNA on sperm cell association. The aim of this work was to evaluate the effect of size, concentration, and structure of exogenous DNA sequence in apoptosis and oncosis induction and the association with spermatozoa. To evaluate the effect of size and concentration of exogenous DNA, sequences of 2.2, 5.5, and 8.5 kb were used at the concentrations of 500 ng (Experiment 1) or 5 × 1011 molecules (Experiment 2). To assess the effect of structure (Experiment 3), 3 sequences of approximately 500 bp were used, containing 65.7% of AT (AT sequence), 46.4% of AT (IN sequence), and 38.6% of AT (GC sequence). To evaluate apoptosis and oncosis in all treatments, sperm cells were incubated with exogenous DNA for 1 h, stained with 2 µL of Yo-Pro (100 µM) for 20 min, and 10 µL of propidium iodide (6 µM) for 10 min, and then analyzed by flow cytometry. To evaluate the transfection rates, sperm cells, after incubation, were separated from non-associated exogenous DNA by 2.5% gel electrophoresis. DNA (genomic and exogenous) was extracted and association rates obtained by real-time PCR. Data were submitted to ANOVA and compared by Tukey's test (P ≤ 0.05). The number of viable, apoptotic, oncosis, or in initial apoptosis/late oncosis spermatozoa incubated with different concentrations or sequences of exogenous DNA did not differ among groups. In Experiment 1, a higher DNA association with sperm cells was observed in the 5.5 kb sequence in comparison with the 2.2 and 8.5 kb. In Experiment 2, the 8.5 kb sequence had the lower association with the spermatozoa in comparison with the 2.2 kb and 5.5 kb. In Experiment 3, there was a higher interaction of the AT sequence when compared to IN and GC sequences. Reference data suggest that big sequences of exogenous DNA have advantages in the interaction with spermatozoa. The present work is the first one to show that the advantage of the big sequences on interaction became a disadvantage in relation to the ability of these cells to internalize this DNA. Table 1. Quantification of different exogenous DNA associated to sperm cells Financial support was provided by FAPESP 03/10234-7 and 03/07456-8.

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Molecular cloning of suppressor of sable, a Drosophila melanogaster transposon-mediated suppressor.

Molecular and Cellular Biology ◽

10.1128/mcb.6.5.1520 ◽

1986 ◽

Vol 6 (5) ◽

pp. 1520-1528 ◽

Cited By ~ 14

Author(s):

D Y Chang ◽

B Wisely ◽

S M Huang ◽

R A Voelker

Keyword(s):

Drosophila Melanogaster ◽

Dna Sequences ◽

Insertion Site ◽

Hybrid Dysgenesis ◽

P Element ◽

Dna Transformation ◽

Wild Type ◽

Induced Mutations ◽

X Ray ◽

Element Insertion

A hybrid dysgenesis-induced allele [su(s)w20] associated with a P-element insertion was used to clone sequences from the su(s) region of Drosophila melanogaster by means of the transposon-tagging technique. Cloned sequences were used to probe restriction enzyme-digested DNAs from 22 other su(s) mutations. None of three X-ray-induced or six ethyl methanesulfonate-induced su(s) mutations possessed detectable variation. Seven spontaneous, four hybrid dysgenesis-induced, and two DNA transformation-induced mutations were associated with insertions within 2.0 kilobases (kb) of the su(s)w20 P-element insertion site. When the region of DNA that included the mutational insertions was used to probe poly(A)+ RNAs, a 5-kb message was detected in wild-type RNA that was present in greatly reduced amounts in two su(s) mutations. By using strand-specific probes, the direction of transcription of the 5-kb message was determined. The mutational insertions lie in DNA sequences near the 5' end of the 5-kb message. Three of the seven spontaneous su(s) mutations are associated with gypsy insertions, but they are not suppressible by su(Hw).

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Expression and Integration of Exogenous DNA Sequences Transfected Into Mammalian Cells

Advanced Research on Animal Cell Technology ◽

10.1007/978-94-009-0875-8_18 ◽

1989 ◽

pp. 261-275

Author(s):

F. Colbere-Garapin ◽

M. L. Ryhiner ◽

A. C. Garapin

Keyword(s):

Dna Sequences ◽

Mammalian Cells ◽

Exogenous Dna

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Novel generation of human satellite DNA-based artificial chromosomes in mammalian cells

Journal of Cell Science ◽

10.1242/jcs.113.18.3207 ◽

2000 ◽

Vol 113 (18) ◽

pp. 3207-3216 ◽

Cited By ~ 1

Author(s):

E. Csonka ◽

I. Cserpan ◽

K. Fodor ◽

G. Hollo ◽

R. Katona ◽

...

Keyword(s):

Dna Sequences ◽

Satellite Dna ◽

Mammalian Cells ◽

Large Scale ◽

De Novo ◽

Mammalian Species ◽

Genetic Material ◽

Exogenous Dna ◽

Artificial Chromosomes ◽

Acrocentric Chromosomes

An in vivo approach has been developed for generation of artificial chromosomes, based on the induction of intrinsic, large-scale amplification mechanisms of mammalian cells. Here, we describe the successful generation of prototype human satellite DNA-based artificial chromosomes via amplification-dependent de novo chromosome formations induced by integration of exogenous DNA sequences into the centromeric/rDNA regions of human acrocentric chromosomes. Subclones with mitotically stable de novo chromosomes were established, which allowed the initial characterization and purification of these artificial chromosomes. Because of the low complexity of their DNA content, they may serve as a useful tool to study the structure and function of higher eukaryotic chromosomes. Human satellite DNA-based artificial chromosomes containing amplified satellite DNA, rDNA, and exogenous DNA sequences were heterochromatic, however, they provided a suitable chromosomal environment for the expression of the integrated exogenous genetic material. We demonstrate that induced de novo chromosome formation is a reproducible and effective methodology in generating artificial chromosomes from predictable sequences of different mammalian species. Satellite DNA-based artificial chromosomes formed by induced large-scale amplifications on the short arm of human acrocentric chromosomes may become safe or low risk vectors in gene therapy.

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Integrative transformation of Candida albicans, using a cloned Candida ADE2 gene.

Molecular and Cellular Biology ◽

10.1128/mcb.6.1.142 ◽

1986 ◽

Vol 6 (1) ◽

pp. 142-149 ◽

Cited By ~ 103

Author(s):

M B Kurtz ◽

M W Cortelyou ◽

D R Kirsch

Keyword(s):

Candida Albicans ◽

Dna Sequences ◽

Southern Hybridization ◽

Sexual Cycle ◽

Dna Transformation ◽

Common Region ◽

Integrative Transformation ◽

Dimorphic Yeast ◽

Ade2 Gene ◽

Yeast Plasmids

Candida albicans is a diploid dimorphic yeast with no known sexual cycle. The development of a DNA transformation system would greatly improve the prospects for genetic analyses of this yeast. Plasmids were isolated from a Candida Sau3A partial library which complements the ade2-1 and ade2-5 mutations in Saccharomyces cerevisiae. These plasmids contain a common region, part of which, when subcloned, produces ade2 complementation. Among the small number of auxotrophs previously isolated in C. albicans, red adenine-requiring mutants had been identified by several groups. In two of these strains, the cloned Candida DNA transformed the mutants to ADE+ at frequencies of 0.5 to 5 transformants per micrograms of DNA. In about 50% of the transformants, plasmid DNA sequences became stably integrated into the host genome and, in the several cases analyzed by Southern hybridization, the DNA was integrated at the site of the ADE2 gene in one of the chromosomal homologs.

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5580859 Delivery of exogenous DNA sequences in a mammal

Biotechnology Advances ◽

10.1016/s0734-9750(97)87877-9 ◽

1997 ◽

Vol 15 (3-4) ◽

pp. 763

Keyword(s):

Dna Sequences ◽

Exogenous Dna

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Small Activating RNAs: Towards the Development of New Therapeutic Agents and Clinical Treatments

Cells ◽

10.3390/cells10030591 ◽

2021 ◽

Vol 10 (3) ◽

pp. 591

Author(s):

Hossein Ghanbarian ◽

Shahin Aghamiri ◽

Mohamad Eftekhary ◽

Nicole Wagner ◽

Kay-Dietrich Wagner

Keyword(s):

Gene Expression ◽

Dna Sequences ◽

Cultured Cells ◽

Exogenous Dna ◽

Evolutionarily Conserved ◽

Rna Activation ◽

Endogenous Genes ◽

Cellular Machinery ◽

Eukaryotic Organisms ◽

Activate Gene

Small double-strand RNA (dsRNA) molecules can activate endogenous genes via an RNA-based promoter targeting mechanism. RNA activation (RNAa) is an evolutionarily conserved mechanism present in diverse eukaryotic organisms ranging from nematodes to humans. Small activating RNAs (saRNAs) involved in RNAa have been successfully used to activate gene expression in cultured cells, and thereby this emergent technique might allow us to develop various biotechnological applications, without the need to synthesize hazardous construct systems harboring exogenous DNA sequences. Accordingly, this thematic issue aims to provide insights into how RNAa cellular machinery can be harnessed to activate gene expression leading to a more effective clinical treatment of various diseases.

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