Anitha Mamillapalli

AAGAG repeat RNA is an essential component of nuclear matrix in Drosophila

Eukaryotic nucleus is functionally as well as spatially compartmentalized and maintains dynamic organization of sub-nuclear bodies. This organization is supported by a non-chromatin nuclear structure called the nuclear matrix. Although the precise molecular composition and ultra-structure of the nuclear matrix is not known, proteins and RNA molecules are its major components and several nuclear matrix proteins have been identified. However, the nature of its RNA component is unknown. Here we show that in Drosophila melanogaster, transcripts from AAGAG repeats of several hundred nucleotide in length are critical constituents of the nuclear matrix. While both the strands of this repeat are transcribed and are nuclear matrix associated, the polypurine strand is predominantly detected in situ. We also show that AAGAG RNA is essential for viability. Our results reveal the molecular identity of a critical RNA component of the nuclear architecture and point to one of the utilities of the repetitive part of the genome that has accumulated in higher eukaryotes.

Developmental proteome dynamics of silk glands in the 5th instar larval stage of Bombyx mori L (CSR2 × CSR4)

UNLABELLED Bivoltine breed of Bombyx mori (B. mori), CSR2×CSR4 is an Indian high yielding silkworm strain. Silk gland proteome of this strain was not studied till now. Methods of improving silk production by chemical approaches have reached saturation and transgenic methods are needed in further to boost silk production. An understanding of proteomic changes during silk gland development helps in designing experiments to enhance silk production by transgenic approaches. The present study reports comprehensive developmental proteomic analysis of CSR2×CSR4, 5th instar whole silk glands. Eighty six unique protein IDs were obtained from the analysis of one hundred and twenty protein spots. Among the identified proteins, majority of the proteins were involved in metabolism (41%) followed by proteins involved in protein homeostasis (30%). Sixty percent of the identified proteins showed dynamic nature by expression analysis from day 1, day 3, day 5 and day 7 gels. In comparison to the published data till now on silk gland proteomics this study reports identification of 20 new proteins from the silk glands for the first time. SIGNIFICANCE The paper reports for the first time proteomic analysis of high yielding silkworm strain of India. The study analyzes whole silk glands to understand the tissue in total during 5th instar development. Lowering fibroin content made us to identify a large number of new proteins which were not reported till now in the silk gland proteome. Proteins which are involved in silk synthesis and release were found to be developmentally regulated. The study identified alanine, serine and glycine tRNA ligases for the first time and also showed their up-regulation on day 7 of 5th instar larval stage. The amino acid repeat of fibroin protein is enriched with the three amino acids, glycine, serine and alanine. The identified proteins could be studied further to understand their functional role in-depth.

Spermidine enhances the silk production by mulberry silkworm.

Abstract Polyamines are ubiquitous low molecular weight polycationic aliphatic amines involved in diverse cellular processes. Spermidine (Spd), a polyamine, has been proved to be crucial for cell survival in various organisms. Our study reports the effect of Spd on the growth of Bombyx mori . Silkworms showed improved silk gland weight and economic parameters in the fifth instar larval stage when treated with different concentrations of Spd, in the range of 25–75 µM. The worms treated with Spd produced 31% more silk when compared with the control worms. Altogether, this study establishes that Spd-treated leaves can be fed into the larvae for better silk production.

Transposable element ‘ roo’ attaches to nuclear matrix of the Drosophila melanogaster

Abstract The genome of eukaryotes is organized into structural units of chromatin loops. This higher order organization is supported by a nuclear skeleton called the nuclear matrix. The genomic DNA associated with the nuclear matrix is called the matrix associated region (MAR). Only a few genome-wide screens have been attempted, although many studies have characterized locusspecific MAR DNA sequences. In this study, a MAR DNA library was prepared from the Drosophila melanogaster Meigen (Diptera: Drosophilidae) genome. One of the sequences identified as a MAR was from a long terminal repeat region of ‘roo’ retrotransposon (roo MAR). Sequence analysis of roo MAR showed its distribution across the D. melanogaster genome. roo MAR also showed high sequence similarity with a previously identified MAR in Drosophila, namely the ‘gypsy’ retrotransposon. Analysis of the genes flanking roo MAR insertions in the Drosophila genome showed that genes were co-ordinately expressed. The results from the present study in D. melanogaster suggest this sequence plays an important role in genome organization and function. The findings point to an evolutionary role of retrotransposons in shaping the genomic architecture of eukaryotes.

Effect of polyamines on mechanical and structural properties of Bombyx mori silk.

Silkworm, Bombyx mori (B. mori) belongs to the Lepidoptera family. The silk produced from this insect, mulberry silk, gained lot of importance as a fabric. Silk is being exploited as a biomaterial due to its surprising strength and biocompatibility. Polyamines (PA) are important cell growth regulators. In the present work the effect of treatment of polyamines, putrescine (Put), spermidine (Spd), and spermine (Spm) on the quantity and quality of silk produced was assessed. Results showed that exogenous feeding of Spd at a concentration of 50 µM increased fiber length significantly. Analysis by Fourier transform infrared (FTIR) on the properties of silk obtained from Spd treated silkworms revealed an increase in percentage of absorption with no difference in peak positions of amide I and amide III groups. Scanning electron microscopy (SEM) revealed an increase in diameter of silk. Further, analysis at molecular level showed an increase in fibroin expression in Spd treated silk glands. However, the Spd treatment showed no significant difference with respect to fibroin to sericin ratio per unit weight of cocoon, silk tenacity, and percent elongation. Thus, the present results show that polyamine treatment would influence silk quality at structural, mechanical, and molecular level in the Bombyx mori, which can be exploited in silk biomaterial production.

Knockdown of Broad-Complex Gene Expression of Bombyx mori by Oligopyrrole Carboxamides Enhances Silk Production

Bombyx mori (B. mori) is important due to its major role in the silk production. Though DNA binding ligands often influence gene expression, no attempt has been made to exploit their use in sericulture. The telomeric heterochromatin of B. mori is enriched with 5′-TTAGG-3′ sequences. These sequences were also found to be present in several genes in the euchromatic regions. We examined three synthetic oligopyrrole carboxamides that target 5′-TTAGG-3′ sequences in controlling the gene expression in B. mori. The ligands did not show any defect or feeding difference in the larval stage, crucial for silk production. The ligands caused silencing of various isoforms of the broad-complex transcription factor and cuticle proteins which resulted in late pupal developmental defects. Furthermore, treatment with such drugs resulted in statistically enhanced cocoon weight, shell weight, and silk yield. This study shows for the first time use of oligopyrrole carboxamide drugs in controlling gene expression in B. mori and their long term use in enhancing silk production.