J. Grifo

A short fluorescence in-situ hybridization (FISH) procedure using fluorochrome and digoxigenin labelled DNA probes was developed for application in human preimplantation embryos in order to analyse the five chromosomes most involved in human aneuploidy (X, Y, 18, 13 and 21). The chromosomes were fluorescent-stained and detected simultaneously in 157 blastomeres from 30 human embryos. Successful FISH analysis was achieved in 93% of the blastomeres. Aberrations for these chromosomes were found in 70% of abnormally developing monospermic embryos. The majority of normally developing monospermic embryos obtained from older patients were also chromosomally abnormal. By analysing all or most of the cells from these embryos, true mosaicism was distinguished from technique failure. Mosaic embryos, polyploid embryos with ploidies as high as 8n, haploid embryos, embryos monosomic for 13/21 and for X, and embryos trisomic for 13/21 and 18, were common in abnormally developing embryos. In contrast, aneuploidy was the main chromosome abnormality found in normally developing monospermic embryos.

A reconstituted reticulocyte translation system originally designed to be deficient in eukaryotic initiation factor 4B (eIF-4B) was used to identify a new activity required for maximal synthesis of rabbit globin. This new activity purifies as a stable, high molecular weight complex by a variety of chromatographic procedures and is termed eIF-4F. The purified globin stimulatory activity also restores translation of capped mRNAs in extracts of poliovirus-infected HeLa cells. Like restoring activity that was obtained as a protein complex by different procedures (Tahara, S. M., Morgan, M. A. and Shatkin, A. J. (1981) J. Biol. Chem. 256, 791-794), eIF-4F includes the 24,000-dalton cap binding protein and major polypeptides of Mr approximately 200,000 and approximately 46,000. The latter component comigrates with eIF-4A by two-dimensional gel electrophoresis and, like eIF-4A, chemically cross-links to the 5'-end of capped mRNA by an ATP-dependent, m7GDP-sensitive reaction. Unlike eIF-4F, cap binding protein of Mr approximately 24,000 isolated by affinity chromatography on m7GDP-Sepharose does not stimulate globin synthesis in the reconstituted system.

Interaction of protein synthesis initiation factors with mRNA has been studied in order to characterize early events in the eukaryotic translation pathway. Individual reovirus mRNAs labeled with 32P in the alpha position relative to the m7G cap and eukaryotic initiation factor (eIF)-4A, -4B, and -4F purified from rabbit reticulocytes were employed. It was found that eIF-4A causes a structural change in mRNA, as evidenced by a nuclease sensitivity test: addition of high concentrations of eIF-4A greatly increase the nuclease sensitivity of the mRNA, suggesting that this factor can melt or "unwind" mRNA structure. ATP is required for this reaction. At low concentrations of eIF-4A, addition of eIF-4B is required for maximal unwinding activity. Thus eIF-4B enhances eIF-4A activity. Addition of eIF-4F also makes the mRNA sensitive to nuclease indicating a similar unwinding role to that of eIF-4A. Stoichiometric comparisons indicate that eIF-4F is more than 20-fold more efficient than eIF-4A in catalyzing this reaction. The unwinding activity of eIF-4F is inhibited by m7GDP, while that of eIF-4A is not. This suggests that eIF-4A functions independent of the 5' cap structure. Our results also suggest that the unwinding activity of eIF-4F is located in the 46,000-dalton polypeptide of this complex, which has shown by others to be similar or identical to eIF-4A.