Regulation of fibrion H gene expression in the posterior silk gland of Bombyx mori.

Abstract

The larval silk glands of Bombyx mori have long served as a model system for investigating developmentally regulated, tissue and stage specific gene expression. The silk glands consist of a pair of long tubular organs divided into anatomically and functionally distinct regions that produce the major classes of silk proteins. Fibroin, the silk fibre protein, is synthesized in the posterior silk gland (PSG), whereas sericin, the glue protein that coats the fibroin, is produced exclusively in the middle silk gland (MSG). The silk gland is fully formed at the end of embryonic development and no further cell division occurs. Approximately 18–19 endomitotic DNA replication cycles occur in the PSG and MSG during larval development. The cells in PSG and MSG grow extremely large, and during the last four days of the 5th instar, when silk protein biosynthesis reaches maximal levels, nearly 50% of the total protein produced by the larva is synthesized in the silk glands. Fibroin H protein (Mr 350,000) has an unusual amino acid composition (Gly 46%; Ala 30%; Ser 12%; Tyr 4%) and is composed of several repetitive, identical peptide units. The mRNA for fibroin H is also unusually long (15–16 kb) and consists of short, repetitive nucleotide sequences. It is transcribed from a single copy gene, regulated by a periodic “turn on and turn off” mechanism. The transcription rate during the feeding stages of the 4th and 5th instars is about 14 molecules/min/gene, but it drops nearly 1000 fold during the moulting period. In the final instar, the PSG shows a remarkable functional adaptation to synthesize high levels of fibroin H protein. The fibroin H gene is transcribed very efficiently, leading to enormous accumulation of fibroin H mRNA. The translational machinery also adapts: there is a dramatic rise in isoaccepting tRNAs for Gly, Ala, Ser and Tyr and in their cognate synthetases, matching the translational demand imposed by fibroin H codons. Other species of poly(A) RNAs and tRNAs decrease quantitatively. At the peak of fibroin H synthesis, more than 80% of PSG polysomes are engaged in translating fibroin H mRNA. Although the fibroin H RNA constitutes the same percentage of total PSG RNA during the feeding stages of the 4th and 5th instars, very little fibroin H protein is synthesized during the 4th instar compared to the 5th. This work therefore investigates the transcriptional and translational regulation of fibroin H gene expression in these two stages (non producing vs. producing stages). ________________________________________ Chapter 1 A literature survey on eukaryotic gene expression regulation and on the molecular biology of silk glands with respect to fibroin, sericin, and tRNA genes. ________________________________________ Chapter 2 - Construction of a B. mori Genomic Library A genomic library of B. mori was constructed in the EcoRI site of the Charon 4A bacteriophage vector. High efficiency in vitro packaging extracts yielded 2.8 × 10 recombinant phage particles. A fibroin H–containing clone was isolated using labelled fibroin H mRNA purified by Sepharose 4B chromatography and confirmed by RNase T fingerprinting. Additional confirmation was obtained by hybridization with first strand cDNA synthesized from poly(A) fibroin H mRNA. Insert size, orientation, and a putative restriction map were prepared. ________________________________________ Chapter 3 - Quantitation of Fibroin H RNA and Protein Fibroin H RNA levels were quantified by gel exclusion HPLC and sucrose gradient centrifugation. Fibroin H RNA comprised similar proportions of total RNA in feeding stages of both instars but degraded rapidly during moulting. Poly(A) containing fibroin H RNA showed no major differences between the instars. Anti fibroin H antibodies were raised, purified, radioiodinated, and used for quantitation. Fibroin H protein levels were 17 fold higher in the 5th instar relative to the 4th. Polysome association studies showed: • only 40% of fibroin H mRNA is polysome bound in the 4th instar, • more than 95% is polysome bound in the 5th instar. Moreover, the 4th instar fibroin H protein levels were far lower than expected even from 40% polysome association-indicating translational regulation. ________________________________________ Chapter 4 - tRNA and tRNA Synthetase Levels A rapid and sensitive HPLC based procedure was developed to quantify individual tRNA species and synthetases. This involved aminoacylation with radiolabelled amino acids, deacylation, derivatisation with phenylisothiocyanate, HPLC separation, and scintillation counting. Results showed: • A dramatic increase in tRNAs for Gly, Ala, Ser, Tyr and their synthetases only in the 5th instar. • No such increase occurred in the 4th instar. Thus, despite polysome association, fibroin H mRNA is inefficiently translated in the 4th instar due to limited availability of the required tRNA species. ________________________________________ Chapter 5 - Modified Base (5 Methylcytosine) in Silk Gland DNA A highly sensitive repeated cycle reverse phase HPLC method was developed to detect 5 methylcytosine (5mC) at levels as low as 0.001 mol%. Base analysis studies revealed: • 5mC is present at low abundance in both PSG and MSG DNA. • Its level increases by 17% (PSG) and 15% (MSG) during the transition from 4th to 5th instar. This increased methylation may correspond to transcriptional inactivation of non silk protein genes.