-
ELife Dec 2022Asynchronous replication of chromosome domains during S phase is essential for eukaryotic genome function, but the mechanisms establishing which domains replicate early...
Asynchronous replication of chromosome domains during S phase is essential for eukaryotic genome function, but the mechanisms establishing which domains replicate early versus late in different cell types remain incompletely understood. Intercalary heterochromatin domains replicate very late in both diploid chromosomes of dividing cells and in endoreplicating polytene chromosomes where they are also underreplicated. SNF2-related factor SUUR imparts locus-specific underreplication of polytene chromosomes. SUUR negatively regulates DNA replication fork progression; however, its mechanism of action remains obscure. Here, we developed a novel method termed MS-Enabled Rapid protein Complex Identification (MERCI) to isolate a stable stoichiometric native complex SUMM4 that comprises SUUR and a chromatin boundary protein Mod(Mdg4)-67.2. Mod(Mdg4) stimulates SUUR ATPase activity and is required for a normal spatiotemporal distribution of SUUR in vivo. SUUR and Mod(Mdg4)-67.2 together mediate the activities of insulator that prevent certain enhancer-promoter interactions and establish euchromatin-heterochromatin barriers in the genome. Furthermore, or ) mutations reverse underreplication of intercalary heterochromatin. Thus, SUMM4 can impart late replication of intercalary heterochromatin by attenuating the progression of replication forks through euchromatin/heterochromatin boundaries. Our findings implicate a SNF2 family ATP-dependent motor protein SUUR in the insulator function, reveal that DNA replication can be delayed by a chromatin barrier, and uncover a critical role for architectural proteins in replication control. They suggest a mechanism for the establishment of late replication that does not depend on an asynchronous firing of late replication origins.
Topics: Animals; Drosophila; DNA-Binding Proteins; Heterochromatin; Drosophila melanogaster; Drosophila Proteins; Euchromatin; Chromatin; DNA Replication
PubMed: 36458689
DOI: 10.7554/eLife.81828 -
Insects Oct 2022One of the most popular tools for species discovery and resolution is the DNA barcode, typically based on the cytochrome oxidase I (COI) gene. However, other non-genic...
One of the most popular tools for species discovery and resolution is the DNA barcode, typically based on the cytochrome oxidase I (COI) gene. However, other non-genic barcodes are available for Diptera. The banding sequence of polytene chromosomes in some dipteran cells, particularly of the larval silk glands, can provide a unique species barcode. We used the sequence of bands to reveal a new species of black fly in the () species group from California, USA. To further characterize the species and provide more integrated taxonomy, we morphologically described all life stages above the egg, formally named the species n. sp., and provided a conventional COI barcode. The COI barcode confirmed the chromosomal and morphological evidence that the species is a new member of the group, and enabled identification of the larva and female, which are structurally similar to those of other species. The chromosomal barcode shows that this species has the most rearranged complement, compared with the eight other North American members of its species group, with up to 12 times the number of fixed rearrangements. Up to six chromosomal rearrangements, including autosomal polymorphisms and sex-linked phenomena, are shared with other members of the group. The most unique and conspicuous chromosomal feature of this new species is a large, pale-staining chromocenter from which the six chromosomal arms radiate. The distribution of this univoltine species in lowland rivers of California's Central Valley could make it vulnerable, given climate change and increasing land development.
PubMed: 36292851
DOI: 10.3390/insects13100903 -
PLoS Genetics Oct 2022Meiosis in males of higher dipterans is achiasmate. In their spermatocytes, pairing of homologs into bivalent chromosomes does not include synaptonemal complex and...
Meiosis in males of higher dipterans is achiasmate. In their spermatocytes, pairing of homologs into bivalent chromosomes does not include synaptonemal complex and crossover formation. While crossovers preserve homolog conjunction until anaphase I during canonical meiosis, an alternative system is used in dipteran males. Mutant screening in Drosophila melanogaster has identified teflon (tef) as being required specifically for alternative homolog conjunction (AHC) of autosomal bivalents. The additional known AHC genes, snm, uno and mnm, are needed for the conjunction of autosomal homologs and of sex chromosomes. Here, we have analyzed the pattern of TEF protein expression. TEF is present in early spermatocytes but cannot be detected on bivalents at the onset of the first meiotic division, in contrast to SNM, UNO and MNM (SUM). TEF binds to polytene chromosomes in larval salivary glands, recruits MNM by direct interaction and thereby, indirectly, also SNM and UNO. However, chromosomal SUM association is not entirely dependent on TEF, and residual autosome conjunction occurs in tef null mutant spermatocytes. The higher tef requirement for autosomal conjunction is likely linked to the quantitative difference in the amount of SUM protein that provides conjunction of autosomes and sex chromosomes, respectively. During normal meiosis, SUM proteins are far more abundant on sex chromosomes compared to autosomes. Beyond promoting SUM recruitment, TEF has a stabilizing effect on SUM proteins. Increased SUM causes excess conjunction and consequential chromosome missegregation during meiosis I after co-overexpression. Similarly, expression of SUM without TEF, and even more potently with TEF, interferes with chromosome segregation during anaphase of mitotic divisions in somatic cells, suggesting that the known AHC proteins are sufficient for establishment of ectopic chromosome conjunction. Overall, our findings suggest that TEF promotes alternative homolog conjunction during male meiosis without being part of the final physical linkage between chromosomes.
Topics: Animals; Male; Drosophila melanogaster; Drosophila; Drosophila Proteins; Polytetrafluoroethylene; Chromosome Segregation; Meiosis; Sex Chromosomes; Chromosome Pairing
PubMed: 36251690
DOI: 10.1371/journal.pgen.1010469 -
PLoS Genetics Oct 2022Chromatin insulators are responsible for orchestrating long-range interactions between enhancers and promoters throughout the genome and align with the boundaries of...
Chromatin insulators are responsible for orchestrating long-range interactions between enhancers and promoters throughout the genome and align with the boundaries of Topologically Associating Domains (TADs). Here, we demonstrate an association between gypsy insulator proteins and the phosphorylated histone variant H2Av (γH2Av), normally a marker of DNA double strand breaks. Gypsy insulator components colocalize with γH2Av throughout the genome, in polytene chromosomes and in diploid cells in which Chromatin IP data shows it is enriched at TAD boundaries. Mutation of insulator components su(Hw) and Cp190 results in a significant reduction in γH2Av levels in chromatin and phosphatase inhibition strengthens the association between insulator components and γH2Av and rescues γH2Av localization in insulator mutants. We also show that γH2Av, but not H2Av, is a component of insulator bodies, which are protein condensates that form during osmotic stress. Phosphatase activity is required for insulator body dissolution after stress recovery. Together, our results implicate the H2A variant with a novel mechanism of insulator function and boundary formation.
Topics: Animals; Chromatin; DNA; Drosophila; Drosophila Proteins; Drosophila melanogaster; Histones; Insulator Elements; Microtubule-Associated Proteins; Nuclear Proteins; Phosphoric Monoester Hydrolases; Polytene Chromosomes
PubMed: 36197938
DOI: 10.1371/journal.pgen.1010396 -
Genetica Feb 2023Polyteny is an effective mechanism for accelerating growth and enhancing gene expression in eukaryotes. The purpose of investigation was to study the genetic variability...
Polyteny is an effective mechanism for accelerating growth and enhancing gene expression in eukaryotes. The purpose of investigation was to study the genetic variability of polyteny degree of giant chromosomes in the salivary glands of Drosophila melanogaster Meig. in relation to the differential fitness of different genotypes. 16 strains, lines and hybrids of fruit flies were studied. This study demonstrates the significant influence of hereditary factors on the level of polytenization of giant chromosomes in Drosophila. This is manifested in the differences between strains and lines, the effect of inbreeding, chromosome isogenization, hybridization, adaptively significant selection, sexual differences, and varying degrees of individual variability of a trait in different strains, lines, and hybrids. The genetic component in the variability of the degree of chromosome polyteny in Drosophila salivary glands was 45.3%, the effect of sex was 9.5%. It has been shown that genetic distances during inbreeding, outbreeding or hybridization, which largely determine the selective value of different genotypes, also affect polyteny patterns. Genetic, humoral, and epigenetic aspects of endocycle regulation, which may underlie the variations in the degree of chromosome polyteny, as well as the biological significance of the phenomenon of endopolyploidy, are discussed.
Topics: Animals; Drosophila melanogaster; Chromosomes; Genotype; Hybridization, Genetic; Salivary Glands
PubMed: 36163579
DOI: 10.1007/s10709-022-00168-4 -
Biology Sep 2022The ultrastructure of antipodal cells of the embryo sac was studied at different stages of differentiation and programmed cell death. The importance of cell function in...
The ultrastructure of antipodal cells of the embryo sac was studied at different stages of differentiation and programmed cell death. The importance of cell function in the antipodal complex is evidenced by the fact that it is fully formed before double fertilization, past the stages of proliferation of three initial cells, and several rounds of genome endoreduplication during differentiation. In this study, we showed that the actively synthesizing organelles, the granular reticulum, and Golgi apparatus, alter their structure during differentiation and death. The polymorphism of the shape of the mitochondria and plastids was demonstrated. For the first time, the actin filaments of the cytoskeleton and numerous multivesicular bodies associated with the plasma membrane were detected in the cytoplasm. The transfer of cytoplasm and organelles between antipodal cells and into the coenocyte of the endosperm was confirmed. DNA breaks and the release of cytochrome c at various stages of death were revealed. To understand the function of the antipodal cells, a quantitative PCR analysis of the expression of wheat genes involved in protective, antistress, and metabolic processes was carried out. We found that gene expression in the antipodal cell fraction was increased compared with that in the whole embryo sac. On the basis of the data, we assume that antipodal cells produce both nutrients and numerous antistress factors that ensure the normal development of the endosperm of the grain, which, in turn, further ensures the development of the embryo.
PubMed: 36138819
DOI: 10.3390/biology11091340 -
Zootaxa May 2022The larva of Clunio marinus Haliday is described based on field and reared material, collected along the coast of Vigo (Spain) and Helgoland (Germany). C. marinus can be...
The larva of Clunio marinus Haliday is described based on field and reared material, collected along the coast of Vigo (Spain) and Helgoland (Germany). C. marinus can be easily distinguished from other known European Clunio species on the basis of some species-specific features found in fourth instar larvae. Comparison with related known Clunio species from Europe with comments on their larval morphology are given. The characteristics of the polytene chromosomes of C.marinus from Vigo are presented. The comparative external morphological analysis of the larvae from the two localities shows a certain differentiation at the external morphological level. The reasons for this differentiation are discussed.
Topics: Animals; Chironomidae; Larva; Polytene Chromosomes; Species Specificity
PubMed: 36095798
DOI: 10.11646/zootaxa.5141.2.4 -
PloS One 2022The ribosomal protein uL11 is located at the basis of the ribosome P-stalk and plays a paramount role in translational efficiency. In addition, no mutant for uL11 is...
The ribosomal protein uL11 is located at the basis of the ribosome P-stalk and plays a paramount role in translational efficiency. In addition, no mutant for uL11 is available suggesting that this gene is haplo-insufficient as many other Ribosomal Protein Genes (RPGs). We have previously shown that overexpression of Drosophila melanogaster uL11 enhances the transcription of many RPGs and Ribosomal Biogenesis genes (RiBis) suggesting that uL11 might globally regulate the level of translation through its transcriptional activity. Moreover, uL11 trimethylated on lysine 3 (uL11K3me3) interacts with the chromodomain of the Enhancer of Polycomb and Trithorax Corto, and both proteins co-localize with RNA Polymerase II at many sites on polytene chromosomes. These data have led to the hypothesis that the N-terminal end of uL11, and more particularly the trimethylation of lysine 3, supports the extra-ribosomal activity of uL11 in transcription. To address this question, we mutated the lysine 3 codon using a CRISPR/Cas9 strategy and obtained several lysine 3 mutants. We describe here the first mutants of D. melanogaster uL11. Unexpectedly, the uL11K3A mutant, in which the lysine 3 codon is replaced by an alanine, displays a genuine Minute phenotype known to be characteristic of RPG deletions (longer development, low fertility, high lethality, thin and short bristles) whereas the uL11K3Y mutant, in which the lysine 3 codon is replaced by a tyrosine, is unaffected. In agreement, the rate of translation decreases in uL11K3A but not in uL11K3Y. Co-immunoprecipitation experiments show that the interaction between uL11 and the Corto chromodomain is impaired by both mutations. However, Histone Association Assays indicate that the mutant proteins still bind chromatin. RNA-seq analyses from wing imaginal discs show that Corto represses RPG expression whereas very few genes are deregulated in uL11 mutants. We propose that Corto, by repressing RPG expression, ensures that all ribosomal proteins are present at the correct stoichiometry, and that uL11 fine-tunes its transcriptional regulation of RPGs.
Topics: Animals; Drosophila Proteins; Lysine; Mutation; Ribosomal Proteins; Transcriptional Activation
PubMed: 35981051
DOI: 10.1371/journal.pone.0273198 -
Cold Spring Harbor Protocols Dec 2022Chromosomes are intricately folded within the cell nucleus and interact with peripheral nuclear proteins. The chromatin architecture has a profound effect on how the...
Chromosomes are intricately folded within the cell nucleus and interact with peripheral nuclear proteins. The chromatin architecture has a profound effect on how the genome is organized. 3D-FISH is a powerful technique that can reveal the structural and functional organization of chromosomes in the nuclear space. Here, we present a protocol for visualizing specific genomic regions in whole-mount paraformaldehyde-fixed cell nuclei of mosquitoes. This protocol was tested in our laboratories and has been showed to be effective and reliable for visualizing genomic regions of various lengths-from 1-kb gene-scale fragments to chromosome-scale segments of DNA.
Topics: Animals; Chromatin; In Situ Hybridization, Fluorescence; Cell Nucleus; Chromosomes; Anopheles
PubMed: 35960625
DOI: 10.1101/pdb.prot107873 -
Cold Spring Harbor Protocols Dec 2022Chromosome visualization is a key step for developing cytogenetic maps and idiograms, analyzing inversion polymorphisms, and identifying mosquito species. Three types of...
Chromosome visualization is a key step for developing cytogenetic maps and idiograms, analyzing inversion polymorphisms, and identifying mosquito species. Three types of chromosomes-polytene, mitotic, and meiotic-are used in cytogenetic studies of mosquitoes. Here, we describe a detailed method for obtaining high-quality polytene chromosome preparations from the salivary glands of larvae and the ovaries of females for mosquitoes. We also describe how to obtain mitotic chromosomes from imaginal discs of fourth-instar larvae and meiotic chromosomes from the testes of male pupae for all mosquitoes. These chromosomes can be used for fluorescence in situ hybridization (FISH), a fundamental technique in cytogenetic research that is used for physical genome mapping, detecting chromosomal rearrangements, and studying chromosome organization.
Topics: Male; Animals; Female; In Situ Hybridization, Fluorescence; Polytene Chromosomes; Chromosomes; Anopheles; Chromosome Mapping; Cytogenetic Analysis; Larva
PubMed: 35960616
DOI: 10.1101/pdb.prot107872