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Classification and Systematics
Published in Jacques Derek Charlwood, The Ecology of Malaria Vectors, 2019
Diptera like Anopheles gambiae have, in some of their cells (e.g., ovarian nurse cells, salivary glands of larvae), massive chromosomes formed by repeated lengthwise multiplication of DNA. These so-called polytene chromosomes contain light and dark banding patterns that are of cytotaxonomic value (Figure 1.13). The A. gambiae genome is organised on three chromosomes: two autosomes and one X/Y sex chromosome. For descriptive purposes, the autosomes are divided into two ‘arms’ at the centromere. The longer arm is referred to as the right arm and the shorter the left arm. Inversions are rearrangements of sections of chromosome turned around between break points so that the linear order of the genes is reversed on that part of the chromosome. The nature of the inversions often makes it possible to decide if specimens belong to the same or different species and to detect natural hybrids between them. The presence of fixed species-specific fixed inversions, particularly in chromosome X, allows the identification of most of the Anopheles gambiae complex members (Coluzzi et al., 1985; Coetzee et al., 2013).
Immunological Studies on the Structure and Function of HMG Proteins
Published in Isaac Bekhor, Carol J. Mirell, C. C. Liew, Progress in Nonhistone Protein Research, 1985
Polytene chromosomes, by virtue of their structure and size, offer a convenient experimental system to study the organization of a chromosomal component. These are essentially large chromosomes in which 1000 or more chromatids are aligned precisely.35 Tightly coiled chromatin regions appear as bands while more loosely packed regions appear as interbands. Furthermore, in these chromosomes, transcriptional activity can be induced in defined genetic loci. Antibodies against a variety of proteins have been used to visualize the location of the proteins by immunofluorescence. Among these is the antibody to RNA polymerase which prominently stains interbands and transcriptionally active puffs.36 Antibodies to histone stain mostly bands, and puffed regions are selectively depleted of immunofluorescence.37,38 This is probably due to steric hindrance of histone antigenic determinants by components found in the transcriptionally active regions.39Chironomus thummi contains a protein which by enzyme-linked immunoassay and by “western” blotting cross-reacts with calf thymus HMG. The distribution of accessible antigenic sites of this protein in C. thummi polytene chromosomes was visualized by immunofluorescence. The results indicate that (1) HMG-1 is distributed in a distinct banding pattern along the entire length of the chromosomes; (2) the banding pattern obtained with fluorescent antibody does not strictly correspond to that observed by phase-contrast microscopy; and (3) the amount of HMG-1 increases and the fluorescent banding pattern changes during the development of the organism. These findings suggest that the protein may be involved in the modulation of the structure of selected loci in the chromosome.39 In transcriptionally active puffs the antigenic determinants of HMG-1 and -2 are more accessible than the determinants of histones. However, the distribution of HMG-1 (i.e., the immunofluorescence pattern) does not correlate with transcriptionally active chromosomal regions nor does it change upon treatment of the chromosomes with NaCl, a treatment which induces transcription as measured by 3H-uridine incorporation.
Comprehensive characterization of a transgene insertion in a highly repetitive, centromeric region of Anopheles mosquitoes
Published in Pathogens and Global Health, 2023
Matteo Vitale, Chiara Leo, Thomas Courty, Nace Kranjc, John B. Connolly, Giulia Morselli, Christopher Bamikole, Roya Elaine Haghighat-Khah, Federica Bernardini, Silke Fuchs
Ovaries at Christopher stage III from 18/33-h half-gravid females were dissected from 7 wild-type and 8 Ag(PMB1) individuals and fixed for 24-h at Room Temperature (RT) in Carnoy’s solution. Polytene chromosome preparations were obtained according to Xia et al. [23]. Chromosome spread quality check was performed using a phase-contrast microscope and then immersed in Liquid Nitrogen. Subsequently, they were dehydrated in a series of ethanol (50%, 70%, 90%, 100%) for 5 minutes each and then allowed to air dry at RT and stored at – 20°C. To perform Fluorescent In Situ Hybridization (FISH) on each chromosome preparation, 800 ng of pBac [3xP3-DsRed]b2eGFP::I-PpoI124L plasmid DNA [11] was labeled with dUTP-Cy5 (GE Healthcare) by nick translation reaction (ROCHE). The reaction mixture was incubated at 15°C for 90 minutes and stopped by heating at 65°C for 10 minutes. PCR reaction (F-TATCGGCTGCAACATCAAAC, R-ACAGAGGTTGTTGAGGAACCA) was used to generate probes from the gene AGAP004670 [24]. DNA probes were precipitated by adding 1/10 volume of 3 M NaAC and 2.5 volume of 100% ethanol and centrifuged at 14,000 g for 20 minutes at 4°C. The Probe was then resuspended in hybridization buffer that was pre-warmed at 37°C. FISH was performed as previously described by Xia et al. [23].
The byssal-producing glands and proteins of the silverlip pearl oyster Pinctada maxima (Jameson, 1901)
Published in Biofouling, 2022
Alessandra Whaite, Anne Klein, Shahida Mitu, Tianfang Wang, Abigail Elizur, Scott Cummins
The P. maxima thread proteome uncovered a homologue of a BR3 protein. Balbiani rings were first discovered through the study of polytene chromosomes of the large, highly specialised salivary gland cells in Chironomus (midge) larvae and are of interest to the broader scientific community as they contain experimentally observable sites of active RNA synthesis (Wieslander and Paulsson 1992; Wieslander 1994). At the larval stage, the polytene chromosomes in the salivary gland cells of C. tentans contains between 8,200–16,400 chromatid copies, allowing for high gene expression in this tissue (Björk and Wieslander 2015). C. tentans rapidly constructs a silk larval tube from this specialised salivary tissue utilising BR3 proteins, which are thought to protect proteins in the gland lumen from forming insoluble silk fibres too soon (Paulsson et al. 1990). The P. maxima BR3-like partial sequence was highly expressed and contains a KAZAL-type serine protease inhibitor domain. Serine proteases can interact with thrombospondin-1 and metalloproteinases; therefore, the BR3-like protein may offer a protective mechanism to the TSP-1 proteins (Rosini et al. 2018). The presence of a BR3 homologue in P. maxima byssus is a novel finding and may offer an interesting avenue for further study, particularly as evidence of holocrine secretion, which is the secretion mechanism reported in Mytilus spp., has not yet been observed in Pinctada spp. (Waite et al. 2004). High expression of an uncharacterised BRICHOS-domain containing protein in the stem region was also an interesting discovery. BRICHOS domains have been shown to engage with and chaperone peptides inclined to form β-sheet structures and potentially protect against amyloid fibril formation (Knight et al. 2013). Understanding the nuance of secretion would guide the evaluation of expression systems for recombinant byssal proteins.