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Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-Cas system has been discovered as an adaptive-immune system in prokaryotes. Microbes like bacteria and archaea use CRISPR-Cas9 as a part of their defense mechanism to ward off the virus and cleave their DNA. Over the past decades, researchers have identified that this simple CRISPR-Cas9 system of bacteria can be utilized to cut any DNA. It is also possible to make precise editing in the genome of almost any organism. This discovery has revolutionized the CRISPR-Cas9 tools and made it one of the most precise gene editing technology known till date. The simple, versatile and programmable nature of CRISPR-Cas9 system 5wthat contains a single guide RNA and Cas9 enzyme, made it an attractive choice for genome editing application. Scientists in the field of molecular biology, genetics and medicine extensively use this transformative technology to study gene regulation and also for treatment of several incurable genetic diseases. Today, CRISPR-Cas9 is the most powerful breakthrough of the century for its immense potential to modulate gene expression in living cells and its application to medicine and human health. Recently, ethical challenges associated with the application of this technology to human health become a hot debate in the scientific community. In this chapter the brief history of development of CRISPR-Cas9 tools and its immense application potential have been discussed.

Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-Cas system has been discovered as an adaptive-immune system in prokaryotes. Microbes like bacteria and archaea use CRISPR-Cas9 as a part of their defense mechanism to ward off the virus and cleave their DNA. Over the past decades, researchers have identified that this simple CRISPR-Cas9 system of bacteria can be utilized to cut any DNA. It is also possible to make precise editing in the genome of almost any organism. This discovery has revolutionized the CRISPR-Cas9 tools and made it one of the most precise gene editing technology known till date. The simple, versatile and programmable nature of CRISPR-Cas9 system 5wthat contains a single guide RNA and Cas9 enzyme, made it an attractive choice for genome editing application. Scientists in the field of molecular biology, genetics and medicine extensively use this transformative technology to study gene regulation and also for treatment of several incurable genetic diseases. Today, CRISPR-Cas9 is the most powerful breakthrough of the century for its immense potential to modulate gene expression in living cells and its application to medicine and human health. Recently, ethical challenges associated with the application of this technology to human health become a hot debate in the scientific community. In this chapter the brief history of development of CRISPR-Cas9 tools and its immense application potential have been discussed.

The transcription factor Gcr1 controls expression of over 75% of the genes in actively growing yeast. Yet, despite its widespread effects, regulation of Gcr1 itself remains poorly understood. Here we show that posttranscriptional Gcr1 regulation is nutrient-dependent. Moreover, GCR1 RNA contains a long, highly conserved intron, which allows the cell to generate multiple RNA and protein isoforms whose levels change upon glucose depletion. Intriguingly, an isoform generated by intron retention is exported from the nucleus, and its translation is initiated from a conserved, intronic translation start site. Expression of gene products from both the spliced and unspliced RNAs is essential, as cells expressing only one isoform cannot adjust their metabolic program in response to environmental changes. Finally, we show that the Gcr1 proteins form dimers, providing an elegant mechanism by which this one gene, through its regulation, can perform the repertoire of transcriptional activities necessary for fine-tuned environmental response.

The primary technical constraint plant scientists face in generating insect resistant transgenic crops with insecticidal Bacillus thuringiensis (Bt) crystal protein (Cry) genes remains failing to generate sufficiently large numbers of effective resistant transgenic plant lines. One possible means to overcome this challenge is through deployment of a Cry toxin gene that contains high levels of insecticidal specific activity for target insect pests. In the present study, we tested this hypothesis using a natural variant of the Cry1Ab toxin under laboratory conditions that possessed increased insecticidal potency against the yellow stem borer (YSB, Scirpophaga incertulus), one of the most damaging rice insect pests. Following adoption of a stringent selection strategy for YSB resistant transgenic rice lines under field conditions, results showed recovery of a significantly higher number of YSB resistant independent transgenic plant lines with the variant cry1Ab gene relative to transgenic plant lines harbouring cry1Ab berliner gene. Structural homology modelling of the variant toxin peptide with the Cry1Aa toxin molecule, circular dichroism spectral analysis, and hydropathy plot analysis indicated that serine substitution by phenylalanine at amino acid position 223 of the Cry1Ab toxin molecule resulted in a changed role for α-helix 7 in domain I of Cry1Ab for enhanced toxicity.

Recent studies have revealed a close relationship between transcription, histone modification, and RNA processing. In fact, genome-wide analyses that correlate histone marks with RNA processing signals raise the possibility that specific RNA processing factors may modulate transcription and help to "write" chromatin marks. Here we show that the nuclear cap binding complex (CBC) directs recruitment of transcription elongation factors and establishes proper histone marks during active transcription. A directed genetic screen revealed that deletion of either subunit of the CBC confers a synthetic growth defect when combined with deletion of genes encoding either Ctk2 or Bur2, a component of the Saccharomyces cerevisiae ortholog of P-TEFb. The CBC physically associates with these complexes to recruit them during transcription and mediates phosphorylation at Ser-2 of the C-terminal domain (CTD) of RNA polymerase II. To understand how these interactions influence downstream events, histone H3K36me3 was examined, and we demonstrate that CBCΔ affects proper Set2-dependent H3K36me3. Consistent with this, the CBC and Set2 have similar effects on the ability to rapidly induce and sustain activated gene expression, and these effects are distinct from other histone methyltransferases. This work provides evidence for an emerging model that RNA processing factors can modulate the recruitment of transcription factors and influence histone modification during elongation.

The potential of insecticidal Vip3Aa toxin peptide of Bacillus thuringiensis (Bt) as a resource for development of lepidopteran insect resistant transgenic crop plants has not yet been fully fathomed. The single mode of protection offered by the insecticidal Vip3Aa toxin against a broad spectrum of lepidopteran insect pests that invade crop field as secondary insect pests, carry definitive significance. However, lack of diversity amongst insecticidal Vip3A toxin towards toxicity for lepidopteran insects is often considered as disadvantage. In order to bring in improvement at this front, search for diversity and protein engineering of the toxin molecule for creation of diversity require to be undertaken in future. In that context, identification of the bioactive core component of Vip3BR toxin peptide of Bt an analogue of Vip3Aa toxin has been accomplished. The core component was found to contain enhanced potency of the insecticidal property 2–3 folds more than the native toxin against four major crop pests.

Alternative pre-mRNA splicing allows dramatic expansion of the eukaryotic proteome and facilitates cellular response to changes in environmental conditions. The Saccharomyces cerevisiae gene SUS1, which encodes a protein involved in mRNA export and histone H2B deubiquitination, contains two introns; non-canonical sequences in the first intron contribute to its retention, a common form of alternative splicing in plants and fungi. Here we show that the pattern of SUS1 splicing changes in response to environmental change such as temperature elevation, and the retained intron product is subject to nonsense-mediated decay. The activities of different splicing factors determine the pattern of SUS1 splicing, including intron retention and exon skipping. Unexpectedly, removal of the 3' intron is affected by splicing of the upstream intron, suggesting that cross-exon interactions influence intron removal. Production of different SUS1 isoforms is important for cellular function, as we find that the temperature sensitivity and histone H2B deubiquitination defects observed in sus1Δ cells are only partially suppressed by SUS1 cDNA, but SUS1 that is able to undergo splicing complements these phenotypes. These data illustrate a role for S. cerevisiae alternative splicing in histone modification and cellular function and reveal important mechanisms for splicing of yeast genes containing multiple introns.

Genes of three naturally occurring monocot mannose-binding lectins from snowdrop (Galanthus nivalis L. agglutinin, GNA), garlic (Allium sativum L. leaf agglutinin, ASAL), and onion (Allium cepaL. agglutinin, ACA) and a recombinant fusion lectin between ASAL and ACA genes were expressed in a bacterial system. The pure and active form of the recombinant lectin peptides were utilized for estimation of their sensitivity potential against feeding nymphs of mustard aphid [Lipaphis erysimi (Kaltenbach)], a major sap-sucking insect pest of Indian mustard [Brassica juncea (L.) Czern.], an oilseed crop. The artificial diet bioassay revealed that ACA and the fusion lectin contained higher toxicity potential than GNA and ASAL. Ectopic expression of these lectins in mustard plants confirmed their protective capacity on the development of the population of aphids on transgenic plants. Based on the strong possibilities that lectins originating from diverse sources would have differential insecticidal potential against different insects, deployment of the appropriate lectin gene figures as crucial in the transgenic approach to protect crop plants against sapsucking insect pests.

Main conclusion Ectopic expression of a deletion mutant (ndv200) ofBacillus thuringiensisvip3BRgene in tobacco plant provided almost complete protection against major crop pests cotton boll worm (Helicoverpa armigera), black cut worm (Agrotis ipsilon) and cotton leaf worm (Spodoptera littoralis). Whereasvip3BRtransgenic tobacco plant failed to protect themselves from these insects and showed resistance towards cotton leaf worm only. An analogous form of the Bacillus thuringiensisvip3Aa insecticidal toxin gene, named vip3BR, was identified and characterized, and exhibited similar attributes to the well-known Vip3Aa toxin. Vip3BR possessed broad-spectrum lepidopteran-specific insecticidal properties effective against most major crop pests of the Indian subcontinent. A Vip3BR toxin protein N-terminal deletion mutant, Ndv200, showed increased insecticidal potency relative to the native toxin, which conferred efficacy against four major crop pests, including cotton boll worm (Helicoverpa armigera), black cut worm (Agrotis ipsilon), cotton leaf worm (Spodoptera littoralis), and rice yellow stem borer (Scirpophaga incertulas). Ligand blot analysis indicated the Ndv200 toxin recognized the same larval midgut receptors as the native Vip3BR toxin, but differed from receptors recognized by Cry1A toxins. In the present study, we tested the prospect of the vip3BR and ndv200 toxin gene as candidate in development of insect-resistant genetically engineered crop plants by generating transgenic tobacco plant. The study revealed that the ndv200 mutant of vip3BR insecticidal toxin gene is a strong and prospective candidate for the next generation of genetically modified crop plants resistant to lepidopteran insects.

Pre-mRNA splicing is a critical step in eukaryotic gene expression, which involves removal of noncoding intron sequences from pre-mRNA and ligation of the remaining exon sequences to make a mature message. Splicing is carried out by a large ribonucleoprotein complex called the spliceosome. Since the first description of the pre-mRNA splicing reaction in the 1970s, elegant genetic and biochemical studies have revealed that the enzyme that catalyzes the reaction, the spliceosome, is an exquisitely dynamic macromolecular machine, and its RNA and protein components undergo highly ordered, tightly coordinated rearrangements in order to carry out intron recognition and splicing catalysis. Studies using the genetically tractable unicellular eukaryote budding yeast (Saccharomyces cerevisiae) have played an instrumental role in deciphering splicing mechanisms. In this chapter, we discuss how yeast genetics has been used to deepen our understanding of the mechanism of splicing and explore the potential for future mechanistic insights using S. cerevisiae as an experimental tool.

The study analyzed the different factors that are responsible for the harassment of women garment workers in Bangladesh. Three garment factories from Mirpur area under Dhaka district were selected purposively where garment factories are available. The sample consisted of 90 women workers taking 30 randomly from each of the three garment factories. Female workers are mostly employed at the lower category of jobs like operator, finishing helper, polyer etc. These jobs are very monotonous in nature. Because of the nature of their jobs, female workers sometimes lose interest in work and become depressed. A large number of female workers received low and irregular wages which create their job dissatisfaction. Only 22 female workers earned salary between Tk. 2700 to Tk. 3000 per month. Female workers are sexually harassed by their co-workers in the factory or by police or by mastans in the street. Communication problem is a major problem faced by most of the female garment workers. A long distance travel is not only physical strenuous but also mentally stressful. Their overtime rate is very low. Long working hours result in a number of illnesses and diseases like headache, eye trouble, ear ache, musculoskeletal pain etc. Women are exploited easily due to lack of technical knowledge and training. The employers do not pay any heed to this exploitation. Keywords: Garment industry; Women workers; Harassment DOI: 10.3329/jbau.v8i2.7940 J. Bangladesh Agril. Univ. 8(2): 291-296, 2010

Pre-messenger RNA splicing is carried out by a large ribonucleoprotein complex called the spliceosome. Despite the striking evolutionary conservation of the spliceosomal components and their functions, controversy persists about the relative importance of splicing in Saccharomyces cerevisiae—particularly given the paucity of intron-containing genes in yeast. Here we show that splicing of one pre-messenger RNA, SUS1, a component of the histone H2B ubiquitin protease machinery, is essential for establishing the proper modification state of chromatin. One protein complex that is intimately involved in pre-mRNA splicing, the yeast cap-binding complex, appears to be particularly important, as evidenced by its extensive and unique genetic interactions with enzymes that catalyze histone H2B ubiquitination. Microarray studies show that cap binding complex (CBC) deletion has a global effect on gene expression, and for ∼20% of these genes, this effect is suppressed when ubiquitination of histone H2B is eliminated. Consistent with this finding of histone H2B dependent effects on gene expression, deletion of the yeast cap binding complex leads to overubiquitination of histone H2B. A key component of the ubiquitin-protease module of the SAGA complex, Sus1, is encoded by a gene that contains two introns and is misspliced when the CBC is deleted, leading to destabilization of the ubiquitin protease complex and defective modulation of cellular H2B levels. These data demonstrate that pre-mRNA splicing plays a critical role in histone H2B ubiquitination and that the CBC in particular helps to establish the proper state of chromatin and proper expression of genes that are regulated at the level of histone H2B ubiquitination.