A molecule of chlorophyll is synthesized from eight molecules of delta-aminolevulinate (DALA), the universal precursor of porphyrins. The light-regulated conversion of glutamate to delta-aminolevulinate in the stroma of greening plastids involves the reduction of glutamate to glutamate-1-semialdehyde and its subsequent transamination. The components performing this conversion have been isolated from barley and Chlamydomonas and separated into three fractions by serial affinity chromatography on Blue Sepharose and haem- or chlorophyllin-Sepharose. The complete reaction can be performed in vitro in a reconstituted assay by combining all three fractions. An RNA is the essential component of the chlorophyllin-Sepharose-bound fraction. By nucleotide sequence analysis, we have now identified this RNA as a chloroplast glutamate acceptor RNA. Glutamate attached by an aminoacyl bond to the 3'-terminal adenosine of this RNA is a substrate for the enzyme(s) which perform the subsequent reactions. This reaction represents a novel role for transfer RNA: participation in the metabolic conversion of its cognate amino acid into another metabolite of low relative molecular mass which subsequently is not used in peptide bond synthesis.

The complete nucleotide sequence of citrus exocortis viroid (CEV, propagated in Gymura) and chrysanthemum stunt viroid (CSV, propagated in Cineraria) has been established, using labelling in vilro and direct RNA sequencing methods and a new screening procedure for the rapid selection of suitable RNA fragments from limited digests.The covalently closed circular single-stranded viroid RNAs consist of 371 (CEV) and 354 (CSV) nucleotides, respectively. As previously shown for potato spindle tuber viroid (PSTV, 359 nucleotides), CEV and CSV also contain a long polypurine sequence. Maximal base-pairing of the established CEV and CSV sequences results in an extended rod-like secondary structure similar to that previously established for PSTV and as predicted from detailed physicochemical studies of all these viroids. Although the three viroid species sequenced to date differ in size and nucleotide sequence, there is 60-73 homology between them. As PSTV, CEV and CSV also contain conserved complementary sequences which are separated from each other in the native secondary structure. We postulate that the resulting 'secondary' hairpins, being formed and observed in vitro during the complex process of thermal denaturation of viroid RNA, must have a vital, although yet unknown, function in vivo. The possible origin and function of viroids are discussed on the basis of the characteristic structural features and of a considerable homology with U l a RNA found for a region highly conserved in the three viroids.

Human somatic cells have essentially no telomerase activity. Telomerase is linked to tumor genesis and is a valuable marker for malignant growth. Extreme paucity of the enzyme neccessitated development of a PCR-based assay, 'telomeric repeat amplification protocol' (TRAP). Unfortunately, this method is not without difficulties. Amplification products are not related to the size of the amplified telomerase products. Furthermore, false positive results can occur, and careful control of reaction conditions is crucial. We analyzed in detail the molecular basis of artifacts. Based on these data, reverse PCR primer was changed and both problems in the TRAP assay were eliminated.

The hypothetical replicase or replicase subunit cistron in the 5′‐proximal part of tobacco mosaic virus (TMV) RNA yields a major 126‐K protein and a minor 183‐K ‘readthrough’ protein in vivo and in vitro. Two natural suppressor tRNAs were purified from uninfected tobacco plants on the basis of their ability to promote readthrough over the corresponding UAG termination codon in vitro. In a reticulocyte lysate the yield of 183‐K readthrough protein increases from ˜10% in the absence of added tobacco plant tRNA up to ˜35% in the case of pure tRNATyr added. Their amino acid acceptance and anticodon sequence (GψA) identifies the two natural suppressor tRNAs as the two normal major cytoplasmic tyrosine‐specific tRNAs. tRNATyr1 has an A:U pair at the base of the TψC stem and an unmodified G10, whereas tRNATyr2 contains a G:C pair in the corresponding location and m2G in position 10. This is the first case that, in a higher eukaryote, the complete structure is known of both the natural suppressor tRNAs and the corresponding viral RNA on which they exert their function. The corresponding codon‐anticodon interaction, which is not in accordance with the wobble hypothesis, and the possible biological significance of the readthrough phenomenon is discussed.

Eukaryotic chromosome termini consist of telomeres, short sequence repeats. According to the telomere hypothesis, DNA replication leads to telomere shortening, resulting in a cellular mitotic clock. Telomerase resets it by telomere synthesis. In mammals with a limited growth phase, telomerase activity in somatic tissues is restricted to stem cell derivatives with high proliferation potential. But other animals, like some fish, grow throughout their life with little senescence. All somatic cells require a high proliferation capacity and telomerase should be active in all cells, irrespective of fish age. Indeed, we detected high telomerase activities in all analyzed organs of rainbow trout (Oncorhynchus mykiss).z 1998 Federation of European Biochemical Societies.