Clostridial apoferredoxin messenger ribonucleic acid. Assay and partial purification.

An assay for Clostridium pasteurianum apoferredoxin messenger ribonucleic acid (mRNA) was developed, based on the synthesis of the protein in vitro. Quantitation of apoferredoxin synthesis was accomplished by trypsinization of the cell-free incubation labeled with 3H- or 14C-labeled amino acids, separation of the products by SDS-urea polyacrylamide gel electrophoresis, and excising and counting the NCS-solubilized gel band corresponding to the unique 52-amino acid tryptic peptide derived from apoferredoxin. Its synthesis was shown to be RNA dependent, and was optimized with respect to several parameters of the in vitro protein-synthesizing system. The specificity of the assay was examined with RNA from Clostridium acidi urici, a related species the ferredoxin of which does not yield the 52-amino acid tryptic peptide, and by the use of [3H]leucine, which is not present in C. pasteurianum apoferredoxin. By these methods, the overestimation of apoferredoxin synthesis due to the comigration of fragments from other in vitro products with the legitimate apoferredoxin-derived peptide could be accounted for. The apoferredoxin mRNA was partially purified by the sequential zonal sucrose gradient centrifugation of total RNA followed by Sephadex G-200 chromatography of the enriched RNA, after which a fraction was obtained in which apoferredoxin mRNA was 20-fold enriched. The enriched RNA fraction can now be used for further purification of the apoferredoxin-coding sequences by cloning procedures.

The N-terminal, dehydrogenase/cyclohydrolase domain of yeast cytoplasmic trifunctional C1-tetrahydrofolate synthase requires the C-terminal, synthetase domain for the catalytic activity in vitro.

The yeast ADE3(1-333) gene which encodes a truncated protein containing the N-terminal 5,10-methylene-tetrahydrofolate (THF) dehydrogenase (D)/5,10-methyl-THF cyclohydrolase (C) domain of cytoplasmic trifunctional C1-THF synthase is able to complement all the phenotypes associated with ade3 mutations in vivo. However, expression of the ADE3(1-333) gene in an ade3 strain does not retain any D activity in vitro. Expression in a yeast ade3 strain of the ADE3(1-333) fused to the Escherichia coli lacZ gene or to the yeast SER2 gene allows detection of D and C activities in vitro. These results indicate that the N-terminal D/C domain of C1-THF synthase requires the C-terminal 10-formyl-THF synthetase domain for stable catalytic activity in vitro.

Judgments of origin and generation effects: comparisons between young and elderly adults.

In 2 experiments, young and elderly adults were required to both read words, and generate words by completing word fragments. Subjects were then required to recognize those words that had been presented earlier; for those words that they recognized they judged whether the items had initially been presented in read or generate form. Generation effects (better memory for words that were generated as compared with words that were read) of similar magnitude were observed for both young and older adults. The older adults were consistently less accurate than the younger adults in their judgments of origin. In addition, the young adults exhibited a bias to respond "read" for these judgments. In contrast, the older adults either exhibited a neutral response bias or were biased to respond "generate." Age-related differences in the encoding or retrieval of information about cognitive operations do not provide a good account of the results. Alternative accounts are described.

Age deficits in recall under optimal study conditions.

Young and older adults studied lists of words under both standard and optimal study conditions for subsequent free recall. Under optimal conditions, the participants studied each word for as long as they wished, were allowed to take notes, and were encouraged to actively use whatever strategies they thought would maximize recall. Both age groups recalled more words under optimal study conditions than under standard conditions, but the improvement was greater for the young adults. This increase in the age-related recall deficit was not due to differences in study time. The results suggest that standard laboratory memory tasks do not overestimate the memory deficits of older adults because of a failure to provide either optimal learning conditions or sufficient study time.

Chemical synthesis of folylpolyglutamates, their reduction to tetrahydro derivatives, and their activity with yeast C1-THF synthase.

Pteroyldi- through -pentaglutamic acids were synthesized through the use of the carbodiimide method, rather than the anhydride method or solid phase synthesis. The compounds were converted to the tetrahydro derivatives by enzymatic reduction. The Km values of the folate coenzymes for yeast C1-THF synthase were determined. The determination of the values for the formyltetrahydrofolate synthetase activity of the multifunctional enzyme was possible through the use of newly devised assay based on the fluorescence properties of the pteridine derivatives. The value for the tetraglutamyl coenzyme derivative was approximately 1000-fold lower than that of the monoglutamyl coenzyme, tetrahydrofolate.

Effects of repetition of mental operations on memory for occurrence and origin.

In two experiments, subjects read or generated items at both encoding and retrieval. At test, they were required to decide whether or not the targets were presented initially (recognition), and if so, whether they were initially read or generated (judgments of origin). Recognition for items that were initially generated was enhanced if they were once again generated at test in the same context, but not if they were generated at test without context. These results confirm that memory for occurrence is facilitated by repetition of the initial encoding operations at retrieval. Generating at test resulted in an increase in "generate" responses both for items that were initially generated and for items that were initially read. Overall, there was a decrease in the accuracy of origin discriminations. It is suggested that, when subjects generate at test, they are likely to mistakenly attribute these just-performed operations to be part of the memory trace for that item.

Priming in episodic memory.

Young and old adults studied related and unrelated word pairs and were given both cued recall and recognition tests. The recognition test required speeded responses to single words. The test order was constructed so that half of the B items from each A-B pair were preceded by its paired A item whereas the other half of the B items were preceded by some other old item. Priming was measured as the difference in reaction time between these two types of items. Significant age differences were found in both recall and recognition accuracy, but young and old adults showed equal amounts of priming. There were significant main effects of relatedness on all three dependent measures, but only in cued recall was there a larger age deficit for unrelated items. The results are inconsistent with an age-related deficit for integrating pairs of words at encoding and suggest, instead, an impairment of effortful retrieval processes.

Aging and recognition failure.

Young and old adults studied lists of A-B paired associates that were subsequently tested for both cued recall and recognition of the B items. Significant age deficits were found for both recall and recognition. The age deficit in recognition was attributed to differences in the effectiveness of the retrieval process in recognition, with no age deficit in the process of evaluating an item's familiarity. Recognition failure, the failure to recognize items that are subsequently recalled, was also observed; older adults showed a higher recognition failure rate than younger adults. These results are discussed within the context of current theories of the recognition failure phenomenon.

Isolation and characterization of the Saccharomyces cerevisiae MIS1 gene encoding mitochondrial C1-tetrahydrofolate synthase.

C1-Tetrahydrofolate synthase is a trifunctional polypeptide found in eukaryotic organisms that catalyzes 10-formyltetrahydrofolate synthetase (EC 6.3.4.3), 5,10-methenyltetrahydrofolate cyclohydrolase (EC 3.5.4.9), and 5,10-methylenetetrahydrofolate dehydrogenase (EC 1.5.1.5) activities. In Saccharomyces cerevisiae, C1-tetrahydrofolate synthase is found in both the cytoplasm and the mitochondria. The gene encoding yeast mitochondrial C1-tetrahydrofolate synthase was isolated using synthetic oligonucleotide probes based on the amino-terminal sequence of the purified protein. Hybridization analysis shows that the gene (designated MIS1) has a single copy in the yeast genome. The predicted amino acid sequence of mitochondrial C1-tetrahydrofolate synthase shares 71% identity with yeast C1-tetrahydrofolate synthase and shares 39% identity with clostridial 10-formyltetrahydrofolate synthetase. Chromosomal deletions of the mitochondrial C1-tetrahydrofolate synthase gene were generated using the cloned MIS1 gene. Mutant strains which lack a functional MIS1 gene are viable and can grow in medium containing a nonfermentable carbon source. In fact, deletion of the MIS1 locus has no detectable effect on cell growth.

Inefficient translation of T7 late mRNA by Bacillus subtilis ribosomes. Implications for species-specific translation.

Bacillus subtilis 30 S subunits inefficiently recognize initiation sites in mRNAs from Gram-negative bacteria, but they are able to efficiently recognize initiation sites in mRNA derived from Gram-positive bacteria. McLaughlin et al. (McLaughlin, J. R., Murray, C. L., and Rabinowitz, J. C. (1981) J. Biol. Chem. 256, 11283-11291) have suggested that B. subtilis ribosomes require a strong Shine-Dalgarno sequence for translation initiation. To test whether this criterion is sufficient to explain the translational specificity of B. subtilis ribosomes, T7 late mRNA, which contains strong Shine-Dalgarno sequences before many of the late genes (Dunn, J. J., and Studier, F. W. (1983) J. Mol. Biol. 166, 477-535), was translated in vitro with both Escherichia coli and B. subtilis ribosomes. The identification of several of the in vitro products upon gel electrophoresis indicated that B. subtilis ribosomes recognize correct translation initiation sites in late T7 mRNA, but they do not translate these products efficiently. Competition experiments demonstrated that late T7 mRNA does not inhibit B. subtilis ribosomal translation of B. subtilis derived mRNA (from the bacteriophage phi 29). It is concluded that strong Shine-Dalgarno sequences may be necessary in B. subtilis translation initiation sites; however, additional determinants of initiation which differ from those found in the translation initiation sites of E. coli mRNAs must exist.