G Arroyo’s research while affiliated with Polytechnic University of Puerto Rico and other places

Within the series of timed differential accumulations of small RNAs we have shown to prelude the synthesis of fibroin in the large ampullate glands of Nephila clavipes (Nc), we are currently directing our attention to the alanine tRNAs. This work reports the subcloning of the members of a tRNAAla gene cluster and the optimization of their transcription in a heterologous cell-free system derived from Bombyx mori (Bm) silkglands. Our data show that the heterologous cell-free system supports the faithful and differential transcription of the individual spider alanine tRNA genes. We are thus making use of the extract to characterize the individual genes with respect to flank-contained regulatory elements through cell-free transcription of gene derivatives. The work has been initiated with pNTA3 because of its high transcriptional activity. Interestingly, the transcription of this gene requires a far upstream sequence, an uncommon modality in tRNA genes.

Our studies show that the making of fibroin by mechanically stimulated spider glands seems to require a series of well-orchestrated gene expressions. Monitoring of the process through time sequence has revealed four transient waves of molecular syntheses. The last and most dramatic of these events is the synthesis of the full-size fibroin product, which is preceded by a wave which generates template RNA by a 60-min interval. The other two events generate small RNAs. Analyses of the first of the small RNA-generating bouts, consistently of higher magnitude than the subsequent one, displays upgrading of 5S RNA, to a higher extent of Ul snRNA, and a dramatic boost in alanine transfer RNA (tRNA) accumulation. This tRNA resolves into two isoforms, one of which is gland-specific and quantitatively correlated to its fibroin-synthesizing activity. The second of these waves serves to optimize the gland’s translational milieu through the differential expression of the tRNAs cognate to the most preponderant amino acids of the gland’s fibroin product in a similar proportion to that in which these appear in the fibroin. Worthy of note is the disproportionate accumulation of alanine-tRNA which is produced primarily within the first wave of small RNA syntheses and which selectively enriches the system with a tissue-specific isoacceptor species in a proportion of 4:1 to its constitutive counterpart. The nucleotide sequence of this isoform endows it with structural features which foster its possible performance in other than elongation functions during the synthesis of fibroin.

In studying the process of protein synthesis of a silk-producing organism we have found that several macromolecules must be synthesized in order for the process to occur. Through time course studies, we have found that small RNAs may play a paramount role in directing the finely orchestrated process. Alanine tRNA, U1 snRNA, and 5S RNA have been identified through Northern blotting as molecules timely and tissue-specific synthesized and upgraded as a prelude activity for the silk being produced.

The large ampullate glands of the orb-web spider, Nephila clavipes provide massive amounts of fibroin throughout the lifetime of the adult female. We have developed methods to culture the glands and manipulate their biosynthetic activity. This has allowed us to monitor a series of molecular events that precede silk production in glands excised from appropriately stimulated animals. In this paper, we demonstrate that prior to the transient dramatic production of fibroin, such glands accumulate large amounts of tRNAs cognate to the abundant amino acids in spider silk. One of these, alanine tRNA, appears to consist of two isoaccepting forms--one constitutive, and the other silkgland specific. Moreover, the silkgland-specific form appears to accumulate preferentially in response to stimulation. This phenomenon of tissue-specific tRNA production appears similar to that found in the silkglands of Bombyx mori, but the spider system has the unique property of permitting manipulation in vitro. Thus, it provides an unusual opportunity to study the mechanism of regulated tRNA synthesis.

The production of silk is a specialized case of the capacity of organisms to produce fibrous proteins. Silks are produced for a variety of life needs, and their production is widespread within the animal kingdom, particularly in the phylum Arthropoda, and more specifically in the classes Insecta and Arachnida. Of the insects, the best-known silk producers are the moths and butterflies, and within the arachnids, the spiders. The best-characterized of the fibroin synthesizers has been the silkworm, Bombyx mori. The system is known for its massive production of silk, late in the last molt or fifth instar.