Incorporation of nonnatural amino acids into proteins by using various four-base codons in an Escherichia coli in vitro translation system.

Incorporation of nonnatural amino acids into proteins is a powerful technique in protein research. Amber suppression has been used to this end, but this strategy does not allow multiple incorporation of nonnatural amino acids into single proteins. In this article, we developed an alternative strategy for nonnatural mutagenesis by using four-base codons. The four-base codons AGGU, CGGU, CCCU, CUCU, CUAU, and GGGU were successfully decoded by the nitrophenylalanyl-tRNA containing the complementary four-base anticodons in an Escherichia coli in vitro translation system. The most efficient four-base decoding was observed for the GGGU codon, which yielded 86% of the full-length protein containing nitrophenylalanine relative to the wild-type protein. Moreover, highly efficient incorporation of two different nonnatural amino acids was achieved by using a set of two four-base codons, CGGG and GGGU. This work shows that the four-base codon strategy is more advantageous than the amber suppression strategy in efficiency and versatility.

[Comparison of predictive methods of Sugi (Cryptomeria) atmospheric pollen counts using mean temperature, solar radiation and male flowering index--an example of Toyama Prefecture].

The previous years mean temperature of July, total solar radiation of July and August and the male flowering index were used to predict atmospheric pollen counts. We examined the adaptability of these predictions and we get following results: 1. Following good harvest years of male flower, atmospheric pollen counts predicted by previous year's mean temperature of July and the total solar radiation of July and August are higher than the actual atmospheric pollen count. 2. Prediction of atmospheric pollen counts by factors within the previous five years are more exact because matured Cryptomeria forests are increasing yearly. 3. There are few errors in prediction by the flowering index because the flower index reflects the actual harvest of male flower.

[Relationships between atmospheric Sugi (Japanese cedar)-pollen counts and indices of climatic conditions].

Atmospheric pollen surveys have been conducted using Durham's standard sampler for 12 years in Toyama Prefecture. The relationships between Sugi (Japanese cedar, Cryptomeria japonica D. Don) pollen and some indices of climatic conditions were analyzed. The results were as follows: 1. Total pollen counts showed a tendency to increase for 12 years, although the variation of the count was relatively large. 2. Significant correlations were found between pollen counts and some indices of the climatic conditions in July of the previous year, such as duration of sunshine, mean daily temperature and daily maximum, as well as minimum temperature. 3. Significant correlation was found between pollen count and duration of sunshine in January and March, and the amount of snowfall in March of the observed year using multiple regression analysis. Pollen counts were shown to be relatively high in years with short sunshine in January and long sunshine and heavy snow in March.

[Characteristics of pollen scattering from male sugi flowers in relation to occurring of symptoms in Japanese cedar pollenosis patients].

Generally, airborne scattering of sugi (Cryptomeria japonica D. Don) pollen begins to be observed coincidentally with the flowering of the male sugi flowers between February and April. However the male sugi flowers disperse a certain amount of pollen in early January, only small levels of which can be detected with a Durham's sampler. In some Japanese cedar pollinosis patients, allergic symptoms have been reported to occur in early January. Among 1,366 Japanese cedar pollinosis patients studied, 265 have experienced allergic symptoms before the first day of pollen scattering as determined with a Durham's sampler. Accordingly, it is suggested that substantial amounts of sugi pollen are scattered from early January, and that clinical symptoms can be induced in highly sensitive Japanese cedar pollinosis patients from early January, even before the flowering of sugi.

[Sugi (Cryptomeria japonica D. Don) pollen scattering out of season].

We investigated the preseasonal blooming conditions of the male flowers of Sugi (Cryptomeria japonica D. Don) which flowers commonly in spring. We also investigated atmospheric Sugi pollen during all seasons for three years from 1988 through 1991 to determine pollen scattering in autumn. Our results showed that a discernible amount of pollen is scattered from the middle of October into December; this scattering would be expected in the following spring. Almost none of the buds of the flowers collected in October and in November bloomed when incubated at temperatures ranging from 6 degrees C to 20 degrees C after low-temperature treatment to arise the blooms from dormancy. However, small numbers of the male flowers from various Sugi trees bloomed and scattered pollen under these conditions. We found that the ratio of the blooming male flowers was one-third or less per blossom. The ratio changed depending on the growth stage and incubation conditions. In conclusion, the density of airborne Sugi pollen scattering is not high in autumn in general. However, the density would be expected to be high enough to induce allergic reactions and clinical symptoms in some areas of Sugi growth, especially in a good harvest year.

[Prediction of the day on which sugi (Cryptomeria japonica D. Don) pollen scattering will begin--from view point of plant physiology].

We investigated the cumulative thermal constant and threshold temperature for the development of sugi (Cryptomeria japonica D. Don) male flowers to predict the day on which pollen scattering would begin. The threshold temperature for the development of sugi male flowers was 0.17 degrees C and the cumulative thermal constant was 184 degrees C/day. Using these results, we determined how long it would take for sugi to be awaken from dormancy and we investigated the meteorological date. Reactivation from dormancy requires 3 days, where the minimum temperature is less than 3 degrees C with a mean temperature of less than 6 degrees C; the following day must have a maximum temperature of more than 10 degrees C, and these conditions must be maintained for at least 2 days. From these results, it is not difficult to predict the day on which sugi pollen scattering will begin.

[Relationships between the pollen productions in sugi (Cryptomeria japonica D. Don) forests and the patterns of atmospheric scattering pollens in Toyama Prefecture--the example of Toyama Prefecture in 1990].

We investigated the geographical distribution of sugi (Cryptomeria Japonica D. Don) forests and the male flower-setting conditions of them in Toyama prefecture. The relationships between our findings and the atmospheric pollen scattering patterns were also studied. The results were as follows. 1. Atmospheric pollen counts and their scattering patterns were fairly easily explained by the geographical distribution of 30-year old sugi forests, the male flower-setting conditions and the first day pollen was scattered in those forests. 2. Total atmospheric counts and maximum sugi pollen peaks were significantly affected by the proximity of sugi forests at altitudes of less than 200 meter. 3. In the atmospheric pollen survey, the detected pollen counts decreased according to the distance between the observation sites and the sugi forests. And few pollen grains were observed from natural sugi forests growing at distances of 20 km to 30 km from the observation sites.

Messenger RNA methylation, translation and degradation in extracts of interferon-treated cells.

Extracts from interferon-treated, not virus infected EAT cells differ in several biochemical characteristics from extracts of untreated cells. Some of these differences are manifested only if the extracts are supplemented with ds RNA and ATP. Thus, in the extracts from interferon-treated cells these supplements activate a protein kinase and an endonuclease activity as well as an inhibitor of the translation of messenger RNA. The effect of the same supplements in extracts of untreated cells is much less pronounced. Other differences between the two types of extracts do not seem to depend on the addition of ds RNA and ATP. These include an impairment of mRNA cap methylation and an inhibition of peptide chain elongation that can be overcome by the addition of tRNA. The treatment of human (HeLa S3) cells with human interferon is manifested in the cell extract similarly to the treatment of EAT cells with mouse interferon. Studies are underway to isolate and characterize the ds RNA activated enzymes and the inhibitors and to establish how the presence of these in extracts from interferon-treated cells can account for the impairment of virus replication by interferon.