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

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

Development ◽

10.1242/dev.99.1.15 ◽

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.

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Towards end-to-end disease prediction from raw metagenomic data

10.1101/2020.10.29.360297 ◽

2020 ◽

Author(s):

Maxence Queyrel ◽

Edi Prifti ◽

Jean-Daniel Zucker

Keyword(s):

Dna Sequences ◽

Real Life ◽

Multiple Instance Learning ◽

Disease Classification ◽

Metagenomic Data ◽

Numerical Representation ◽

Metagenomic Sequencing ◽

Sequencing Data ◽

End To End ◽

Bioinformatics Workflows

AbstractAnalysis of the human microbiome using metagenomic sequencing data has demonstrated high ability in discriminating various human diseases. Raw metagenomic sequencing data require multiple complex and computationally heavy bioinformatics steps prior to data analysis. Such data contain millions of short sequences read from the fragmented DNA sequences and are stored as fastq files. Conventional processing pipelines consist multiple steps including quality control, filtering, alignment of sequences against genomic catalogs (genes, species, taxonomic levels, functional pathways, etc.). These pipelines are complex to use, time consuming and rely on a large number of parameters that often provide variability and impact the estimation of the microbiome elements. Recent studies have demonstrated that training Deep Neural Networks directly from raw sequencing data is a promising approach to bypass some of the challenges associated with mainstream bioinformatics pipelines. Most of these methods use the concept of word and sentence embeddings that create a meaningful and numerical representation of DNA sequences, while extracting features and reducing the dimentionality of the data. In this paper we present an end-to-end approach that classifies patients into disease groups directly from raw metagenomic reads: metagenome2vec. This approach is composed of four steps (i) generating a vocabulary of k-mers and learning their numerical embeddings; (ii) learning DNA sequence (read) embeddings; (iii) identifying the genome from which the sequence is most likely to come and (iv) training a multiple instance learning classifier which predicts the phenotype based on the vector representation of the raw data. An attention mechanism is applied in the network so that the model can be interpreted, assigning a weight to the influence of the prediction for each genome. Using two public real-life datasets as well a simulated one, we demonstrated that this original approach reached very high performances, comparable with the state-of-the-art methods applied directly on processed data though mainstream bioinformatics workflows. These results are encouraging for this proof of concept work. We believe that with further dedication, the DNN models have the potential to surpass mainstream bioinformatics workflows in disease classification tasks.

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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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Growth, Metamorphosis and Energy Conversion in the Larvae of the Prawn, Palaemon Serratus

Journal of the Marine Biological Association of the United Kingdom ◽

10.1017/s0025315400046427 ◽

1969 ◽

Vol 49 (1) ◽

pp. 77-96 ◽

Cited By ~ 43

Author(s):

M. R. Reeve

Keyword(s):

Energy Conversion ◽

Larval Growth ◽

Energy Conversion Efficiency ◽

Variable Number ◽

Larval Stages ◽

Length Increase ◽

History Of ◽

The Mean ◽

The Individual ◽

Very High

The life-history of the larva of the prawn Palaemon serratus (Pennant) was studied in the laboratory and found to comprise a variable number of stages, dependent on feeding level and other factors. Larval moulting frequency was relatively constant at 3-day intervals, and could be followed in a population by daily examination for exuvia moult-casts. Length increase at each moult progressed arithmetically, and the highest mortalities occurred during metamorphosis. Consumption of Anemia nauplii by each larva rose from 30 to 400 per day. Populations of isolated animals (i.e. when cannibalism was excluded) were inspected daily and the exact number and duration of larval stages determined. Survival through metamorphosis was very high (over 85%) at both high and low feeding levels, although at the latter the larvae passed through more stages with more erratic duration times. At the end of the experiment, when all the larvae had metamorphosed, the mean dry weights at the high and low feeding levels were 7.7 and o.6 mg respectively. Larval stages could not be positively identified morphologically after stage III unless the history of the individual in question was known. It is suggested that the number of larval stages in the sea may also be very variable and difficult to determine.Energy-conversion efficiency was calculated from food uptake measurements and larval growth estimations, and was found to be 50–60% during most of the larval life, falling to 35% when the early post-larvae were taken into account.

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Larval and Early Post-Larval Development of Arctica Islandica

Journal of the Marine Biological Association of the United Kingdom ◽

10.1017/s0025315400070314 ◽

1982 ◽

Vol 62 (4) ◽

pp. 745-769 ◽

Cited By ~ 34

Author(s):

R. A. Lutz ◽

R. Mann ◽

J. G. Goodsell ◽

M. Castagna

Keyword(s):

Larval Development ◽

Growth Rates ◽

Larval Growth ◽

Ammonium Hydroxide ◽

Culture Conditions ◽

Dilute Solution ◽

Arctica Islandica ◽

Larval Stages ◽

Experimental Laboratory ◽

Rearing Techniques

Mature eggs were stripped from ripe adult specimens of Arctica islandica and exposed to a dilute solution of ammonium hydroxide for various lengths of time before addition of stripped sperm. Larval and early post-larval stages were cultured under experimental laboratory conditions using standard bivalve rearing techniques. Larval cultures were maintained at various controlled temperatures ranging from 8·5 to 14·5 °C. Minimum time to settlement was 32 days at a temperature of approximately 13 °C; at temperatures between 8·5 and 10·0 °C, settlement was not observed until approximately 55 days after fertilization. Larval growth rates were significantly faster at temperatures between 11·0 and 145 °C than at temperatures between 8°C. Morphometry of the larval shell and morphology of the larval hinge apparatus were independent of larval growth rates and experimental culture conditions.

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A systematic literature review on the effects of mycotoxin exposure on insects and on mycotoxin accumulation and biotransformation

Mycotoxin Research ◽

10.1007/s12550-021-00441-z ◽

2021 ◽

Author(s):

K. Niermans ◽

A.M. Meyer ◽

E.F. Hoek-van den Hil ◽

J.J.A. van Loon ◽

H.J. van der Fels-Klerx

Keyword(s):

Aflatoxin B1 ◽

Animal Feed ◽

Larval Growth ◽

Insect Species ◽

Comprehensive Overview ◽

Agricultural Industry ◽

Growth And Survival ◽

Safety Issues ◽

Larval Stages ◽

Published Evidence

AbstractNovel protein sources for animal feed are needed, and the use of insects as feed ingredient is explored. The insect production sector offers opportunities for a circular and sustainable approach to feed production by upgrading waste or side streams into high-quality proteins. However, potential food or feed safety issues should be studied in advance. Mycotoxins, such as aflatoxin B1, are natural contaminants commonly found in agricultural crops and have proven to be detrimental to the agricultural industry, livestock, and human health. This systematic review aims to provide a comprehensive overview of the published evidence on effects of mycotoxin exposure on insect growth and survival, mycotoxin accumulation within the insect body, and metabolization of various mycotoxins by insects. The review includes 54 scientific articles published in the past 55 years, in total covering 32 insect species. The main findings are the following: (1) Insects of the order Coleoptera show lower mortality after exposure to aflatoxin B1 when compared to Lepidoptera and Diptera; (2) effects of mycotoxins on larval growth and survival are less detrimental in later larval stages; (3) accumulation of mycotoxins was low in most insect species; (4) mycotoxins are metabolized within the insect body, the degree of which depends on the particular mycotoxin and insect species; (5) cytochrome P450s are the main family of enzymes involved in biotransformation of mycotoxins in some insect species. Results of this review support an optimistic outlook for the use of mycotoxin-contaminated waste streams as substrate for insect rearing.

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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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