Origin of chromosomal abnormalities in nuclear transplants—A reevaluation of nuclear differentiation and nuclear equivalence in amphibians

Berardino, Marie A. Di; Hoffner, Nancy J.

doi:10.1016/0012-1606(70)90094-1

Cited by 71 publications

(12 citation statements)

References 21 publications

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“…12) even when replication is unfinished. Any slight inhibition of DNA synthesis by thioredoxin m would lead to incompletely replicated chromosomes, and these would be fragmented at mitosis (22) with the loss of non-centromere-containing pieces. After a few rounds of slightly incomplete replication followed by fragmentation, chromosomal DNA may be largely eliminated.…”

Section: Discussionmentioning

confidence: 99%

Spinach thioredoxin m inhibits DNA synthesis in fertilized Xenopus eggs.

Hartman

Buchanan

et al. 1993

Proc. Natl. Acad. Sci. U.S.A.

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A role for thioredoxin in metazoan DNA synthesis has been assessed by injecting rapidly dividing Xenopus eggs with purified heterologous thioredoxins, which might act as inhibitors if they were to replace resident thioredoxins in some but not all reaction steps. Of 10 tested proteins, spinach chloroplast thioredoxin m is the most potent inhibitor. Eggs cleave and produce cells lacking nuclei. DNA synthesis is severely reduced. Development arrests before gastrulation. In egg extracts, thioredoxin m inhibits incorporation of radioactive dCTP into DNA of sperm nuclei and M13 phage. Inhibition exceeds 90% when thioredoxin m and M13 DNA are preincubated together. The data support the interpretation that thioredoxins normally participate in initiation of metazoan DNA synthesis.Thioredoxins are a widely distributed group of proteins that mediate both redox and nonredox reactions in prokaryotic and eukaryotic cells (1)(2)(3)(4). In the latter capacity, thioredoxin functions as an essential subunit of phage T7 DNA polymerase (5, 6) and in the maturation of DNA filamentous phages Fl and M13 (7,8 (12), and each was injected with 30 nl of the specified thioredoxin in TB at the stock concentrations indicated in the figure legends and tables or with TB (buffer) alone 40-60 min postfertilization-that is, prior to first cleavage. Embryos were allowed to develop in MMR/5 medium at 21°C (see ref.12 for composition of MMR medium). The progress of development was scored at various intervals. Arrested blastulae were identified as eggs that had cleaved to numerous cells but did not form a blastopore and did not initiate gastrulation. Arrested gastrulae were identified as those that initiated gastrulation but failed to close the blastopore (large yolk plug remaining) and did not initiate neurulation. Arrested neurulae closed the blastopore and raised neural folds but did not complete closure of the folds or differentiate specific tissues such as eyes or muscle. DNA Staining and Microscopic Analysis of Nuclei. Eggswere injected and allowed to develop in MMR/5 medium at 21°C for 8 hr to reach the midblastula transition (13). They were fixed in 4% formaldehyde in MMR/5 for 1 hr. Animal caps were cut off and transferred to MMR/5 containing Hoechst dye 33258 (bisbenzimide), flattened under a coverslip, and observed with Zeiss fluorescence optics. Preparation of Xenopus Sperm Nuclei and Egg Extracts.Xenopus sperm nuclei were prepared by the method ofLohka and Masui (14) and were demembranated by the inclusion of 400 pg of lysolecithin per ml of sperm heads (=106 sperm heads per ml). Extracts from unfertilized Xenopus eggs were also prepared by published methods (14, 15), with a final centrifugation at 10,000 x g for 5 min in a microcentrifuge at a 900 angle. DNA Replication by Sperm Nuclei. Egg extract (20 I1) was added to 2 ,l of sperm nuclei, 1 1.l of [a-32P]

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Section: Discussionmentioning

confidence: 99%

Spinach thioredoxin m inhibits DNA synthesis in fertilized Xenopus eggs.

Hartman

Buchanan

et al. 1993

Proc. Natl. Acad. Sci. U.S.A.

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“…In contrast, the cell cycles of adult somatic cell nuclei of the same animals are all 24 h or more in length. Therefore, the first cleavage of the recipient cytoplasm occurs before the karyokinetic events of the donor nuclei are complete, which can result in a variety of chromosomal aberrations, including ploidy mosaicism, where cells with abnormal karyotypes are present in the resultant nuclear transplanted animal (Di Berardino & Hoffner 1970;Di Berardino 1979, 1997.…”

Section: Introductionmentioning

confidence: 99%

Diploidized eggs reprogram adult somatic cell nuclei to pluripotency in nuclear transfer in medaka fish (Oryzias latipes)

et al. 2007

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Reprogramming of adult somatic cell nuclei to pluripotency has been unsuccessful in non-mammalian animals, primarily because of chromosomal aberrations in nuclear transplants, which are considered to be caused by asynchrony between the cell cycles of the recipient egg and donor nucleus. In order to normalize the chromosomal status, we used diploidized eggs by retention of second polar body release, instead of enucleated eggs, as recipients in nuclear transfer of primary culture cells from the caudal fin of adult green fluorescent protein gene (GFP) transgenic medaka fish (Oryzias latipes). We found that 2.7% of the reconstructed embryos grew into adults that expressed GFP in various tissues in the same pattern as in the donor fish. Moreover, these fish were diploid, fertile and capable of passing the marker gene to the next generation in Mendelian fashion. We hesitate to call these fish 'clones' because we used non-enucleated eggs as recipients; in effect, they may be chimeras consisting of cells derived from diploid recipient nuclei and donor nuclei. In either case, fish adult somatic cell nuclei were reprogrammed to pluripotency and differentiated into a variety of cell types including germ cells via the use of diploidized recipient eggs.

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“…4). This was probably due to damage (possibly random) O-nu 1-nu 2-nu sustained in the first nuclear transfers as shown by Di Berardino and Hoffner [7]. At that time I was subject to considerable criticism.…”

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confidence: 98%

“…Extensive chromosome damage can arise in somatic cell nuclei transplanted to amphibian eggs [7] McGrath and Solter…”

mentioning

confidence: 99%

Nuclear transplantation, the conservation of the genome, and prospects for cell replacement

Gurdon

2017

The FEBS Journal

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Initial nuclear transplantation experiments in Xenopus eggs provided the first evidence for the conservation of the genome after cellular differentiation. This Discovery‐in‐Context Review recounts the early experiments that led to successful nuclear transfer in amphibians and the establishment of totipotency of a differentiated cell and shows how these discoveries paved the way for similar cloning experiments in other organisms.

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Origin of chromosomal abnormalities in nuclear transplants—A reevaluation of nuclear differentiation and nuclear equivalence in amphibians

Cited by 71 publications

References 21 publications

Spinach thioredoxin m inhibits DNA synthesis in fertilized Xenopus eggs.

Spinach thioredoxin m inhibits DNA synthesis in fertilized Xenopus eggs.

Diploidized eggs reprogram adult somatic cell nuclei to pluripotency in nuclear transfer in medaka fish (Oryzias latipes)

Nuclear transplantation, the conservation of the genome, and prospects for cell replacement

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