Most animals display internal and/or external left-right asymmetry. Several mechanisms for left-right asymmetry determination have been proposed for vertebrates and invertebrates but they are still not well characterized, particularly at the early developmental stage. The gastropods Lymnaea stagnalis and the closely related Lymnaea peregra have both the sinistral (recessive) and the dextral (dominant) snails within a species and the chirality is hereditary, determined by a single locus that functions maternally. Intriguingly, the handedness-determining gene(s) and the mechanisms are not yet identified. Here we show that in L. stagnalis, the chiral blastomere arrangement at the eight-cell stage (but not the two- or four-cell stage) determines the left-right asymmetry throughout the developmental programme, and acts upstream of the Nodal signalling pathway. Thus, we could demonstrate that mechanical micromanipulation of the third cleavage chirality (from the four- to the eight-cell stage) leads to reversal of embryonic handedness. These manipulated embryos grew to 'dextralized' sinistral and 'sinistralized' dextral snails-that is, normal healthy fertile organisms with all the usual left-right asymmetries reversed to that encoded by the mothers' genetic information. Moreover, manipulation reversed the embryonic nodal expression patterns. Using backcrossed F(7) congenic animals, we could demonstrate a strong genetic linkage between the handedness-determining gene(s) and the chiral cytoskeletal dynamics at the third cleavage that promotes the dominant-type blastomere arrangement. These results establish the crucial importance of the maternally determined blastomere arrangement at the eight-cell stage in dictating zygotic signalling pathways in the organismal chiromorphogenesis. Similar chiral blastomere configuration mechanisms may also operate upstream of the Nodal pathway in left-right patterning of deuterostomes/vertebrates.

Although substantial progress has been made recently in understanding the establishment of left-right asymmetry in several organisms, little is known about the initial step for any embryo. In gastropods, left-right body handedness is determined by an unknown maternally inherited single gene or genes at closely linked loci and is associated with the sense of spiral cleavage in early embryos. Contrary to what has been believed, we show that temporal and spatial cytoskeletal dynamics for the left- and right-handed snails within a species are not mirror images of each other. Thus, during the third cleavage of Lymnaea stagnalis, helical spindle inclination (SI) and spiral blastomere deformation (SD) are observed only in the dominant dextral embryos at metaphase-anaphase, whereas in the recessive sinistral embryos, helicity emerges during the furrow ingression. Actin depolymerization agents altered both cleavages to neutral. Further, we found a strong genetic linkage between the handedness-specific cytoskeletal organization and the organismal handedness, using backcrossed F4 congenic animals that inherit only 1/16 of dextral strain-derived genome either with or without the dextrality-determining gene(s). Physa acuta, a sinistral-only gastropod, exhibits substantial SD and SI levotropically. Thus, cytoskeletal dynamics have a crucial role in determination of body handedness with further molecular, cellular, and evolutionary implications.

Several human hereditary neuromuscular disease genes are associated with the expansion of CTG or CGG triplet repeats. The DNA syntheses of CTG triplets ranging from 17 to 180 and CGG repeats from 9 to 160 repeats in length were studied in vitro. Primer extensions using the Klenow fragment of DNA polymerase I, the modified T7 DNA polymerase (Sequenase), or the human DNA polymerase ␤ paused strongly at specific loci in the CTG repeats. The pausings were abolished by heating at 70°C. As the length of the triplet repeats in duplex DNA, but not in single-stranded DNA, was increased, the magnitude of pausings increased. The location of the pause sites was determined by the distance between the site of primer hybridization and the beginning of the triplet repeats. CGG triplet repeats also showed similar, but not identical, patterns of pausings. These results indicate that appropriate lengths of the triplets adopt a non-B conformation(s) that blocks DNA polymerase progression; the resultant idling polymerase may catalyze slippages to give expanded sequences and hence provide the molecular basis for this non-Mendelian genetic process. These mechanisms, if present in human cells, may be related to the etiology of certain neuromuscular diseases such as myotonic dystrophy and Fragile X syndrome.

Long CTG triplet repeats which are associated with several human hereditary neuromuscular disease genes are stabilized in ColEl-derived

Early investigations into the interaction between Escherichia coli glutamyl-tRNA synthetase (GluRS) and tRNAGlu have implicated the modified nucleoside 5-[(methylamino)methyl]-2-thiouridine in the first position of the anticodon as an important contact for efficient aminoacylation. However, the experimental methods employed were not sufficient to determine whether the interaction was dependent on the presence of the modification or simply involved other anticodon loop-nucleotides, now occluded from interaction with the synthetase. Unmodified E. coli tRNA(Glu), derived by in vitro transcription of the corresponding gene, is a poor substrate for GluRS, exhibiting a 100-fold reduction in its specificity constant (kcat/KM) compared to that of tRNA(Glu) prepared from an overproducing strain. Through the use of recombinant RNA technology, we created several hybrid tRNAs which combined sequences from the in vitro transcript with that of the native tRNA, resulting in tRNA molecules differing in modified base content. By in vitro aminoacylation of these hybrid tRNA molecules and of tRNAs with base substitutions at positions of nucleotide modification, we show conclusively that the modified uridine at position 34 in tRNA(Glu) is required for efficient aminoacylation by E. coli GluRS. This is only the second example of a tRNA modification acting as a positive determinant for interaction with its cognate aminoacyl-tRNA synthetase.