Distribution, expression and germ line transmission of exogenous DNA sequences following microinjection into Xenopus laevis eggs

Development ◽  
1987 ◽  
Vol 99 (1) ◽  
pp. 15-23
Author(s):  
L.D. Etkin ◽  
B. Pearman
Keyword(s):  
Xenopus Laevis ◽  
Dna Sequences ◽  
Copy Number ◽  
Germ Line ◽  
Gene Promoter ◽  
Early Gene ◽  
Mosaic Pattern ◽  
Exogenous Dna ◽  
Gene Coding ◽  
Germ Line Transmission

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.

scholarly journals Elimination of Foreign DNA during Somatic Differentiation in Tetrahymena thermophila Shows Position Effect and Is Dosage Dependent

2005 ◽  
Vol 4 (2) ◽  
pp. 421-431 ◽  
Author(s):  
Yifan Liu ◽  
Xiaoyuan Song ◽  
Martin A. Gorovsky ◽  
Kathleen M. Karrer
Keyword(s):  
Transposable Elements ◽  
Dna Sequences ◽  
Copy Number ◽  
Tetrahymena Thermophila ◽  
Germ Line ◽  
Position Effect ◽  
Dna Elimination ◽  
Internal Eliminated Sequences ◽  
Somatic Genome ◽  
Foreign Dna

ABSTRACT In the ciliate Tetrahymena thermophila, approximately 15% of the germ line micronuclear DNA sequences are eliminated during formation of the somatic macronucleus. The vast majority of the internal eliminated sequences (IESs) are repeated in the micronuclear genome, and several of them resemble transposable elements. Thus, it has been suggested that DNA elimination evolved as a means for removing invading DNAs. In the present study, bacterial neo genes introduced into the germ line micronuclei were eliminated from the somatic genome. The efficiency of elimination from two different loci increased dramatically with the copy number of the neo genes in the micronuclei. The timing of neo elimination is similar to that of endogenous IESs, and they both produce bidirectional transcripts of the eliminated element, suggesting that the deletion of neo occurred by the same mechanism as elimination of endogenous IESs. These results indicate that repetition of an element in the micronucleus enhances the efficiency of its elimination from the newly formed somatic genome of Tetrahymena thermophila. The implications of these data in relation to the function and mechanism of IES elimination are discussed.

scholarly journals Germ-line transmission of transgenes in Xenopus laevis

1999 ◽  
Vol 96 (25) ◽  
pp. 14389-14393 ◽  
Author(s):  
N. Marsh-Armstrong ◽  
H. Huang ◽  
D. L. Berry ◽  
D. D. Brown
Keyword(s):  
Xenopus Laevis ◽  
Germ Line ◽  
Germ Line Transmission

Denaturing High Performance Liquid Chromatography and Bioinformatics - Two Modern Tools for Extracellular Superoxide Dismutase (SOD3) Gene Promoter Analysis

2008 ◽  
Vol 59 (7) ◽  
Author(s):  
Corina Samoila ◽  
Alfa Xenia Lupea ◽  
Andrei Anghel ◽  
Marilena Motoc ◽  
Gabriela Otiman ◽  
...  
Keyword(s):  
High Performance Liquid Chromatography ◽  
Transcription Factors ◽  
Liquid Chromatography ◽  
Dna Sequences ◽  
High Performance ◽  
Zinc Finger Protein ◽  
High Capacity ◽  
Gene Promoter ◽  
Experimental Approaches ◽  
Myeloid Zinc Finger

Denaturing High Performance Liquid Chromatography (DHPLC) is a relatively new method used for screening DNA sequences, characterized by high capacity to detect mutations/polymorphisms. This study is focused on the Transgenomic WAVETM DNA Fragment Analysis (based on DHPLC separation method) of a 485 bp fragment from human EC-SOD gene promoter in order to detect single nucleotide polymorphism (SNPs) associated with atherosclerosis and risk factors of cardiovascular disease. The fragment of interest was amplified by PCR reaction and analyzed by DHPLC in 100 healthy subjects and 70 patients characterized by atheroma. No different melting profiles were detected for the analyzed DNA samples. A combination of computational methods was used to predict putative transcription factors in the fragment of interest. Several putative transcription factors binding sites from the Ets-1 oncogene family: ETS member Elk-1, polyomavirus enhancer activator-3 (PEA3), protein C-Ets-1 (Ets-1), GABP: GA binding protein (GABP), Spi-1 and Spi-B/PU.1 related transcription factors, from the Krueppel-like family: Gut-enriched Krueppel-like factor (GKLF), Erythroid Krueppel-like factor (EKLF), Basic Krueppel-like factor (BKLF), GC box and myeloid zinc finger protein MZF-1 were identified in the evolutionary conserved regions. The bioinformatics results need to be investigated further in others studies by experimental approaches.

Fas gene promoter –670 polymorphism in gynecological cancer

2006 ◽  
Vol 16 (Suppl 1) ◽  
pp. 179-182 ◽  
Author(s):  
M. Ueda ◽  
Y. Terai ◽  
K. Kanda ◽  
M. Kanemura ◽  
M. Takehara ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Cervical Cancer ◽  
Cancer Patients ◽  
Germ Line ◽  
Gynecological Cancer ◽  
Gene Promoter ◽  
Single Nucleotide ◽  
Increased Risk ◽  
Significant Difference ◽  
Germ Line Polymorphism

Single-nucleotide polymorphism at −670 of Fas gene promoter (A/G) was examined in a total of 354 blood samples from normal healthy women and gynecological cancer patients. They consisted of 95 normal, 83 cervical, 108 endometrial, and 68 ovarian cancer cases. Eighty-three patients with cervical cancer had statistically higher frequency of GG genotype and G allele than 95 controls (P= 0.0353 and 0.0278, respectively). There was no significant difference in the genotype or allele prevalence between control subjects and endometrial or ovarian cancer patients. The Fas −670 GG genotype was associated with an increased risk for the development of cervical cancer (OR = 2.56, 95% CI = 1.08–6.10) compared with the AA genotype. The G allele also increased the risk of cervical cancer (OR = 1.60, 95% CI = 1.05–2.43) compared with the A allele. Germ-line polymorphism of Fas gene promoter −670 may be associated with the risk of cervical cancer in a Japanese population.

Micronuclear DNA sequences from Tetrahymena do not confer mitotic stability on ARS plasmids in Saccharomyces

Genome ◽  
1988 ◽  
Vol 30 (5) ◽  
pp. 690-696 ◽  
Author(s):  
Wendy H. Horsfall ◽  
Ronald E. Pearlman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Sequences ◽  
Copy Number ◽  
Tetrahymena Thermophila ◽  
Assay System ◽  
Mitotic Stability ◽  
Yeast Saccharomyces Cerevisiae ◽  
Genomic Libraries ◽  
Ade2 Gene

Genomic libraries containing micronuclear DNA sequences from Tetrahymena thermophila have been constructed in a vector containing ARS1, SUP11, and ura3 sequences from the yeast Saccharomyces cerevisiae. When transformed into a strain of S. cerevisiae carrying a suppressible ochre mutation in the ade2 gene, viable transformants are obtained only if the transforming plasmid is maintained at a copy number of one or two per cell. Mitotic segregation of the plasmid is easily assessed in a colour assay of transformants. Using this assay system, we showed that micronuclear DNA from Tetrahymena does not contain sequences that confer mitotic stability on yeast ARS-containing plasmids; i.e., sequences that function analogously to yeast centromere sequences. One transformant was analyzed that carries Tetrahymena sequences that maintain the copy number of the ARS plasmid at one or two per cell. However, these sequences do not confer mitotic stability on the transformants and they confer a phenotype in this assay similar to that of the REP3 gene of the yeast 2 μm plasmid.Key words: mitotic stability, centromere, Tetrahymena, Saccharomyces.

Insertion and excision of Caenorhabditis elegans transposable element Tc1

1988 ◽  
Vol 8 (2) ◽  
pp. 737-746
Author(s):  
D Eide ◽  
P Anderson
Keyword(s):  
Amino Acid ◽  
Caenorhabditis Elegans ◽  
Transposable Element ◽  
Dna Sequences ◽  
Germ Line ◽  
Consensus Sequence ◽  
Somatic Cells ◽  
Chain Gene ◽  
Imprecise Excision ◽  
Insertion Sites

The transposable element Tc1 is responsible for most spontaneous mutations that occur in Caenorhabditis elegans variety Bergerac. We investigated the genetic and molecular properties of Tc1 transposition and excision. We show that Tc1 insertion into the unc-54 myosin heavy-chain gene was strongly site specific. The DNA sequences of independent Tc1 insertion sites were similar to each other, and we present a consensus sequence for Tc1 insertion that describes these similarities. We show that Tc1 excision was usually imprecise. Tc1 excision was imprecise in both germ line and somatic cells. Imprecise excision generated novel unc-54 alleles that had amino acid substitutions, amino acid insertions, and, in certain cases, probably altered mRNA splicing. The DNA sequences remaining after Tc1 somatic excision were the same as those remaining after germ line excision, but the frequency of somatic excision was at least 1,000-fold higher than that of germ line excision. The genetic properties of Tc1 excision, combined with the DNA sequences of the resulting unc-54 alleles, demonstrated that excision was dependent on Tc1 transposition functions in both germ line and somatic cells. Somatic excision was not regulated in the same strain-specific manner as germ-line excision was. In a genetic background where Tc1 transposition and excision in the germ line was not detectable, Tc1 excision in the soma still occurred at high frequency.

Germ line transmission of an inactive N-myc allele generated by homologous recombination in mouse embryonic stem cells

1990 ◽  
Vol 10 (12) ◽  
pp. 6755-6758
Author(s):  
B R Stanton ◽  
S W Reid ◽  
L F Parada
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Homologous Recombination ◽  
Embryonic Development ◽  
Cell Lines ◽  
Germ Line ◽  
Es Cells ◽  
Embryonic Stem ◽  
Es Cell ◽  
Germ Line Transmission

We have disrupted one allele of the N-myc locus in mouse embryonic stem (ES) cells by using homologous recombination techniques and have obtained germ line transmission of null N-myc ES cell lines with transmission of the null N-myc allele to the offspring. The creation of mice with a deficient N-myc allele will allow the generation of offspring bearing null N-myc alleles in both chromosomes and permit study of the role that this proto-oncogene plays in embryonic development.

High-frequency germ line gene conversion in transgenic mice

1992 ◽  
Vol 12 (6) ◽  
pp. 2545-2552
Author(s):  
J R Murti ◽  
M Bumbulis ◽  
J C Schimenti
Keyword(s):  
Gene Conversion ◽  
Dna Sequences ◽  
High Frequency ◽  
Germ Line ◽  
Gene Families ◽  
Histochemical Staining ◽  
Evolutionary Significance ◽  
Evolutionary Divergence ◽  
Lacz Gene ◽  
Male Gametes

Gene conversion is the nonreciprocal transfer of genetic information between two related genes or DNA sequences. It can influence the evolution of gene families, having the capacity to generate both diversity and homogeneity. The potential evolutionary significance of this process is directly related to its frequency in the germ line. While measurement of meiotic inter- and intrachromosomal gene conversion frequency is routine in fungal systems, it has hitherto been impractical in mammals. We have designed a system for identifying and quantitating germ line gene conversion in mice by analyzing transgenic male gametes for a contrived recombination event. Spermatids which undergo the designed intrachromosomal gene conversion produce functional beta-galactosidase (encoded by the lacZ gene), which is visualized by histochemical staining. We observed a high incidence of lacZ-positive spermatids (approximately 2%), which were produced by a combination of meiotic and mitotic conversion events. These results demonstrate that gene conversion in mice is an active recombinational process leading to nonparental gametic haplotypes. This high frequency of intrachromosomal gene conversion seems incompatible with the evolutionary divergence of newly duplicated genes. Hence, a process may exist to uncouple gene pairs from frequent conversion-mediated homogenization.

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