Spinach thioredoxin m inhibits DNA synthesis in fertilized Xenopus eggs.

Abstract

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]

Cytoplasmic Thioredoxin Reductase Is Essential for Embryogenesis but Dispensable for Cardiac Development

“…Several reports indicate that the Txn/Txnrd1 systemeither directly or through substrates such as Ref-1 and peroxiredoxins-participates in the regulation of transcription, replication, and DNA repair (3,16). Whether the mammalian Txn/Txnrd system provides reducing equivalents for the synthesis of deoxyribonucleotides by ribonucleotide reductase, as shown for Escherichia coli (21), Saccharomyces cerevisiae (25), and Xenopus laevis (14), is still being debated.…”

Thioredoxins: structure and function in plant cells

“…Human thioredoxin has also been reported to regulate the DNA binding of transcription factors (Schenk et al, 1994), and a thioredoxin-linked mechanism seems to be involved in the control of hormone and interleukin receptors and in signal transduction chains (Tagaya et al, 1989;SantaColoma, Grasso & Reichert, 1991;Boniface & Reichert, 1990;Iwata et al, 1995;Patel, Zhang & Block, 1996). There are also many reports indicating that thioredoxin plays important roles in embryogenesis, cell division and the cell cycle (Hartman et al, 1993;Natsuyama et al, 1993;Salz et al, 1994). In general, thioredoxin is strongly overproduced in stress conditions, as observed in oxidative stress in yeast (Kuge & Jones, 1994).…”

Thioredoxin is Essential for Rhodobacter Sphaeroides Growth by Aerobic and Anaerobic Respiration

“…Indeed, thioredoxin is required for normal DNA synthesis in Saccharomyces cereuisiae (Muller, 1991(Muller, , 1994) via a redox-dependent function (Muller, 1995) and is involved in the earliest embryonic divisions in both Drosophila (Salz et al, 1994) and Xenopus (Hartman et al, 1993). Other sites of thioredoxin action are suggested by its redox regulation of the mammalian transcription factors NF-KB (Toledano & Leonard, 1991 ;Sorachi et al, 1992;Okamoto et al, 1992;Schenk et al, 1994) and AP-1 (Abate et al, 1990;Schenk et al, 1994) , and of the translational activator modulating the synthesis of photosystem I1 reaction centre D1 protein in Chlamydomonas reinhardtii (Danon & May field, 1994).…”