The fat body cell-free system for tissue-specific transcription of plasma protien gene ofBombyx mori

Abstract

A nuclear extract was prepared for the larval fat body of the silkworm, Bombyx mori, and a homologous in vitro system was developed for the transcription of major plasma protein gene of B.mori. The gene for SP1, a storage protein of B.mori, and adenovirus 2 major late (AdML) gene were faithfully transcribed under relatively high template concentrations in the nuclear extract prepared from the fat body of female fifth instar larvae. Complete inhibition of gene transcription by a low concentration of alpha-amanitin indicated that the reaction is catalyzed by RNA polymerase II. At low template concentration (0.6 nM) the fat body nuclear extract transcribed the homologous SP1 gene with high efficiency, while AdML gene and larval cuticle protein gene were only barely transcribed in the same extract. The SP1 gene deleted upstream of the TATA box sequence showed little effect on transcription, whereas mutations that destroy TATA sequence totally abolished the gene transcription. These results suggested that the core promoter region of SP1 gene spanning between positions -44 and +16 is essential for the fat body specific transcription in vitro.

Suppressive activity of a viral histone H4 against two host chromatin remodelling factors: lysine demethylase and SWI/SNF

“…Fat bodies of fourth instar NP larvae were homogenized and used for isolation of nuclear extract (NE) according to a published method (Mine et al, 1995). Briefly, NE (500 µg in 400 µl) was added to 50 µg (100 µl) of recombinant CpBV-H4 (rCpBV-H4) protein in 500 µl of 100 mM PBS (pH 7.4).…”

Establishment of a Novel In Vivo Sex-Specific Splicing Assay System To Identify a trans-Acting Factor That Negatively Regulates Splicing of Bombyx mori dsx Female Exons

“…Briefly, the cells were suspended in five packed cell volumes of buffer C (31) and allowed to swell on ice for 3 min. Nuclei were pelleted and resuspended in 2.2 packed cell volumes of buffer BЈ (31). For lysis, the nuclei were drawn up in a syringe and rapidly pushed through a 26-gauge needle.…”

“…Extraction of nuclei was carried out on ice for 30 min with constant mixing. After extraction, nuclei were pelleted and the supernatant was dialyzed against buffer DB (31). The dialyzed nuclear protein extracts were then frozen in liquid N 2 and stored at Ϫ80°C.…”

Biosynthesis of major plasma proteins in the primary culture of fat body cells from the silkworm, Bombyx mori

“…Each 4 pmol of SP1 primer 228 (5′-TATACTCCTTGGCGATCTCC) or 30K6G1 primer 174 (5′-CGGTCTCGTATTCACCAATG) was end-labeled with [γ-32 P]ATP using T4 polynucleotide kinase. Primer extension was carried out using 3 µg of total RNA and each 5′ end-labeled primer (1×10 5 cpm) as described by Mine et al (1995). The synthesized cDNA was separated on 6% acrylamide-7 M urea gel, and radioactivity was detected by autoradiography.…”

“…Northern blot analyses of the fat body RNA have demonstrated that the biosyntheses of these proteins are regulated in a stage-, tissue-, and sex-dependent manner at the level of gene transcription in fat body cells Sakai et al 1988;Sakurai et al 1988;Fujii et al 1989). Recently, an efficient cell-free transcription system was developed from the nuclear extract of the larval fat body, which preferentially transcribed a homologous storage protein gene in a promoter specific manner (Mine et al 1995).…”