Pericentromeric Region Of Chromosome Research Articles

A D e Novo Supernumerary Ring Chromosome 1 Causes B-Cell Acute Lymphoblastic Leukemia in Monozygotic Twins Due to Independent and Partially Convergent Evolutionary Trajectories

Introduction B-cell acute lymphoblastic leukemia (B-ALL) is the most common pediatric cancer. In recent years, our understanding of B-ALL biology has benefited from genomic and transcriptomic analyses that identify driver alterations in 98% of patients. In addition, prior studies of monozygotic twins, who both developed B-ALL, have provided insight into the developmental timing and natural history of the disease, as well as the identification of novel mechanisms of leukemogenesis. Herein we study a case of monozygotic twins, who developed B-ALL (Table 1). We report the characterization of a de novo supernumerary ring chromosome 1 (SRC1) as an initiator event that followed independent but partially convergent clonal trajectories in each twin. Thus, SRC1 resulted in genomic rearrangements of MEF2D in both twins but targeted distinct fusion gene partners ( BCL9 versus ARNT). To our knowledge, this study represents the first description of MEF2D-ARNT as an event involved in B-ALL, and the association of SRC1 Syndrome with leukemia. Methods We performed integrative whole genome sequencing (WGS) and RNAseq analysis on B-ALL samples at diagnosis and compared this to WGS data from minimal residual disease negative bone marrow aspirates. Coverage levels for tumors and matched normals were: Twin A=104 & 59; Twin B=81 & 53. Tumor purity estimations were 0.80 in Twin A and 0.31 in Twin B. For RNAseq, expression profile efficiencies were 61% and 36% respectively. Comprehensive variant calling was performed using the Isabl platform (Medina et al, 2020) to map all putative somatic and germline events. ALLCatchR (Beder et. al, 2022) was used for molecular B-ALL subtype allocation in 26 samples from our pediatric cohort of patients including the twins (age range 1-23), and MutationTimeR (Gerstung et al, 2020) to calculate the relative timing of copy number gains. All research studies were approved by NYU and MSKCC Institutional Review Boards. Results The tumor mutational burdens for each leukemia sample were 0.86 for Twin A and 0.47 mutations/Mb for Twin B, and they showed independent alterations in oncogenic genes (Table 1). The only B-ALL driver that both twins had partially in common was an intrachromosomal translocation resulting in MEF2D fusions, but each with a distinct partner gene ( BCL9 versus ARNT) which was confirmed by RNAseq (supporting reads: Twin A=48, Twin B=107) and PCR assays (Fig. 1). Each sample demonstrated enhanced transcription levels of MEF2D compared to our reference B-ALL cohort (transcripts per million: Twin A=48.0, Twin B=77.4, median=27.7, sd=19.9), and were transcriptionally classified as MEF2D-subtype by the ALLCatchR classifier. These results functionally supported the role of MEF2D-ARNT as a new oncogenic event. Analysis of germline variants showed a tandem duplication flanking the pericentromeric region of chromosome 1 in both twins (1:120,163,074-157,714,717) where MEF2D BCL9and ARNT are localized, that had been previously identified as SRC1 in only one of them by constitutional genotyping (Table 1). Cell fractions in normal and tumor tissues were: Twin A=14.8% & 50%, Twin B=8.6% & 26.5%. PCR interrogation of parental germline DNA failed to identify SRC1, suggesting this to be a de novo event in the affected twins (Fig. 1). We compared the relative timepoint in which the SRC1 took place in each sample using MutationalTimeR, but we were only able to obtain results in Twin A due to a higher mutational burden (number of variants in SRC1: Twin A=44, Twin B=13). As expected, the tandem duplication that formed the SRC1 was identified as an early mutational event (confidence interval of relative time of appearance: 0-0.4). Conclusions Our results identify SRC1 as the common initiating genomic event leading to MEF2D fusion driven B-ALL in both twins. These findings support a model in which a tandem duplication around a pericentromeric area was an initiating event which caused the scission and circularization of DNA without the loss of genetic material. This led to a de novo acquisition of a SRC1, that after independent but partially convergent clonal trajectories gave rise to 2 distinct oncogenic MEF2D fusion driven leukemias in each twin . Moreover, ARNT is a novel partner gene to MEF2D which has not been previously described. This is the first report of B-ALL association with SRC1 Syndrome, which emphasises the utility of the study of monozygotic twins to identify novel alterations underlying B-ALL.

Evolution of the spinach sex-linked region within a rarely recombining pericentromeric region.

Sex chromosomes have evolved independently in many different plant lineages. Here, we describe reference genomes for spinach (Spinacia oleracea) X and Y haplotypes by sequencing homozygous XX females and YY males. The long arm of the 185 Mb chromosome 4 carries a 13 Mb X-linked region (XLR) and 24.1 Mb Y-linked region (YLR), of which 10 Mb is Y-specific. We describe evidence that this reflects insertions of autosomal sequences creating a "Y duplication region" or "YDR" whose presence probably directly reduces genetic recombination in the immediately flanking regions, although both the X and Y Sex-Linked Regions are within a large pericentromeric region of chromosome 4 that recombines rarely in meiosis of both sexes. Sequence divergence estimates using synonymous sites indicate that YDR genes started diverging from their likely autosomal progenitors about 3 MYA, around the time when the flanking YLR stopped recombining with the XLR. These flanking regions have a higher density of repetitive sequences in the YY than the XX assembly and include slightly more pseudogenes compared with the XLR, and the YLR has lost about 11% of the ancestral genes, suggesting some degeneration. Insertion of a male-determining factor would have caused Y-linkage across the entire pericentromeric region, creating physically small, highly recombining, terminal pseudo-autosomal regions. These findings provide a broader understanding of the origin of sex chromosomes in spinach.

UroVysionTM Fluorescence In Situ Hybridization in Urological Cancers: A Narrative Review and Future Perspectives.

UroVysionTM is a fluorescence in situ hybridization assay that was developed for the detection of bladder cancer (UC accounted for 90%) in urine specimens. It consists of fluorescently labeled DNA probes to the pericentromeric regions of chromosomes 3, 7, 17 and to the 9p21 band location of the P16 tumor suppressor gene, which was approved by the Food and Drug Administration (FDA) in 2001 and 2005, respectively, for urine detection in patients with suspected bladder cancer and postoperative recurrence monitoring. Furthermore, recent studies also demonstrated that U-FISH was useful for assessing superficial bladder cancer patients' response to Bacillus Calmette-Guérin therapy and in detecting upper tract urothelial carcinoma. Therefore, positive U-FISH was well known to urologists as a molecular cytogenetic technique for the detection of UC. However, with the continuous enrichment of clinical studies at home and abroad, U-FISH has shown a broader application space in the detection of various urinary primary tumors and even metastatic tumors. This review focuses on summarizing the research status of U-FISH in UC, non-urothelial carcinoma and metastatic tumor, so as to strengthen urologists' more comprehensive understanding of the application value of U-FISH and better complete the accurate diagnosis and treatment of urological cancers.

The evolutionary patterns of barley pericentromeric chromosome regions, as shaped by linkage disequilibrium and domestication.

SUMMARYThe distribution of recombination events along large cereal chromosomes is uneven and is generally restricted to gene‐rich telomeric ends. To understand how the lack of recombination affects diversity in the large pericentromeric regions, we analysed deep exome capture data from a final panel of 815 Hordeum vulgare (barley) cultivars, landraces and wild barleys, sampled from across their eco‐geographical ranges. We defined and compared variant data across the pericentromeric and non‐pericentromeric regions, observing a clear partitioning of diversity both within and between chromosomes and germplasm groups. Dramatically reduced diversity was found in the pericentromeres of both cultivars and landraces when compared with wild barley. We observed a mixture of completely and partially differentiated single‐nucleotide polymorphisms (SNPs) between domesticated and wild gene pools, suggesting that domesticated gene pools were derived from multiple wild ancestors. Patterns of genome‐wide linkage disequilibrium, haplotype block size and number, and variant frequency within blocks showed clear contrasts among individual chromosomes and between cultivars and wild barleys. Although most cultivar chromosomes shared a single major pericentromeric haplotype, chromosome 7H clearly differentiated the two‐row and six‐row types associated with different geographical origins. Within the pericentromeric regions we identified 22 387 non‐synonymous SNPs, 92 of which were fixed for alternative alleles in cultivar versus wild accessions. Surprisingly, only 29 SNPs found exclusively in the cultivars were predicted to be ‘highly deleterious’. Overall, our data reveal an unconventional pericentromeric genetic landscape among distinct barley gene pools, with different evolutionary processes driving domestication and diversification.

21. A case of supernumerary ring chromosome 20 involving the gene ASXL1 resulting in brain abnormalities

Reports in the medical literature of supernumerary ring chromosome 20 are rare. Previous studies of duplication and triplication involving the pericentromeric regions of chromosome 20 have shown that carriers present with various clinical features that may include developmental delay, subtle facial dysmorphisms, and abnormalities of the brain, skull and hands. The gene ASXL1 has been implicated in the pathogenicity of reported copy number gains. Conversely, heterozygous loss-of-function variants in ASXL1 are associated with Borhing-Opitz syndrome, which has overlapping clinical features with gains involving this gene.

Comparative Karyotype Analysis of Parasitoid Hymenoptera (Insecta): Major Approaches, Techniques, and Results.

A comprehensive review of main approaches, techniques and results of the chromosome study of parasitic wasps is given. In this group, the haploid chromosome number ranges from n = 3 to 23. Distribution of parasitic wasp species by the chromosome number is bimodal, with two obvious modes at n = 6 and 11. Karyotype analysis based on routinely stained preparations of mitotic chromosomes can be used to identify members of taxonomically complicated parasitoid taxa and to distinguish between them. Morphometric study effectively reveals subtle differences between similar chromosome sets of parasitic wasps. If combined with meiotic analysis and/or cytometric data, information on mitotic karyotypes can highlight pathways of the genome evolution in certain parasitoid taxa. C- and AgNOR-banding as well as staining with base-specific fluorochromes detected important interspecific differences within several groups of parasitic wasps. Fluorescence in situ hybridization (FISH) is successfully used for physical mapping of various DNA sequences on parasitoid chromosomes. These techniques demonstrate that heterochromatic segments are usually restricted to pericentromeric regions of chromosomes of parasitic wasps. Haploid karyotypes carrying one or two nucleolus organizing regions (NORs) are the most frequent among parasitoid Hymenoptera. In combination with chromosome microdissection, FISH could become a powerful tool exploring the genome evolution of parasitic wasps. Perspectives of the comparative cytogenetic study of parasitoid Hymenoptera are outlined.

Chromosomal Diversification in Ancistrus Species (Siluriformes: Loricariidae) Inferred From Repetitive Sequence Analysis.

The Ancistrus genus has extensive chromosomal diversity among species, including heteromorphic sex chromosomes occurrence. However, studies have been shown that chromosomal diversity may still be underestimated. Repetitive sequences represent a large part of eukaryotic genomes, associated with mechanisms of karyotypic diversification, including sex chromosomes evolution. This study analyzed the karyotype diversification of two Ancistrus species (Ancistrus sp. 1 and Ancistrus sp. 2) from the Amazon region by classical and molecular chromosomal markers. Conventional chromosome bands and fluorescence in situ hybridization using probes 18S and 5S rDNA, besides (CA)n, (CG)n, (GA)n, (CAC)n, (CAG)n, (CAT)n, (GAA)n, (GAC)n, (TAA)n, and (TTAGGG)n in tandem repeats were determined on the karyotypes. Ancistrus sp. 1 and Ancistrus sp. 2 presented karyotypes with 2n = 38 (20 m + 14sm+4st, XX/XY) and 2n = 34 (20 m + 14sm, without heteromorphic sex chromosomes), respectively. Robertsonian rearrangements can explain the diploid number difference. C-bands occurred in pericentromeric regions in some chromosomes, and a single 18S rDNA locus occurred in both species. The 5S rDNA showed variation in the number of loci between species karyotypes, suggesting the occurrence of unstable sites and rearrangements associated with these sequences in Ancistrus. The microsatellite mapping evidenced distinct patterns of organization between the two analyzed species, occurring mainly in the sex chromosomes in Ancistrus sp. 1, and in the centromeric and pericentromeric regions of chromosomes m/sm in Ancistrus sp. 2. These data shows the extensive chromosomal diversity of repetitive sequences in Ancistrus, which were involved in Robertsonian rearrangements and sex chromosomes differentiation.

DNA Environment of Centromeres and Non-Homologous Chromosomes Interactions in Mouse.

Although the pericentromeric regions of chromosomes that are enriched in tandemly repeated satellite DNA represent a significant part of eukaryotic genomes, they remain understudied, which is mainly due to interdisciplinary knowledge gaps. Recent studies suggest their important role in genome regulation, karyotype stability, and evolution. Thus, the idea of satellite DNA as a junk part of the genome has been refuted. The integration of data regarding molecular composition, chromosome behaviour, and the details of the in situ organization of pericentromeric regions is of great interest. The objective of this work was a cytogenetic analysis of the interactions between pericentromeric regions from non-homologous chromosomes in mouse spermatocytes using immuno-FISH. We analysed two events: the associations between centromeric regions of the X chromosome and autosomes and the associations between the centromeric regions of the autosomal bivalents that form chromocenters. We concluded that the X chromosome forms temporary synaptic associations with different autosomes in early meiotic prophase I, which can normally be found until the pachytene–diplotene, without signs of pachytene arrest. These associations are formed between the satellite-DNA-rich centromeric regions of the X chromosome and different autosomes but do not involve the satellite-DNA-poor centromeric region of the Y chromosome. We suggest the hypothetical model of X chromosome competitive replacement from such associations during synaptic correction. We showed that the centromeric region of the X chromosome in association remains free of γH2Ax-dependent chromatin inactivation, while the Y chromosome is completely inactivated. This finding highlights the predominant role of associations between satellite DNA-rich regions of different chromosomes, including the X chromosome. We suppose that X-autosomal transient associations are a manifestation of an additional synaptic disorder checkpoint. These associations are normally corrected before the late diplotene stage. We revealed that the intense spreading conditions that were applied to the spermatocyte I nuclei did not lead to the destruction of stretched chromatin fibers of elongated chromocenters enriched in satellite DNA. The tight associations that we revealed between the pericentromeric regions of different autosomal bivalents and the X chromosome may represent the basis for a mechanism for maintaining the repeats stability in the autosomes and in the X chromosome. The consequences of our findings are discussed.

Prenatal Diagnosis of Small Supernumerary Marker Chromosome 10 by Array-Based Comparative Genomic Hybridization and Microdissected Chromosome Sequencing.

Interpreting the clinical significance of small supernumerary marker chromosomes (sSMCs) in prenatal diagnosis is still an urgent problem in genetic counselling regarding the fate of a pregnancy. We present a case of prenatal diagnosis of mosaic sSMC(10) in a foetus with a normal phenotype. Comprehensive cytogenomic analyses by array-based comparative genomic hybridization (aCGH), sSMC microdissection with next-generation sequencing (NGS) of microdissected library, fluorescence in situ hybridization (FISH) with locus-specific and telomere-specific DNA probes and quantitative real-time PCR revealed that sSMC(10) had a ring structure and was derived from the pericentromeric region of chromosome 10 with involvement of the 10p11.21-p11.1 and 10q11.21-q11.23 at 1.243 Mb and 7.173 Mb in size, respectively. We observed a difference in the length of sSMC(10) between NGS data of the DNA library derived from a single copy of sSMC(10), and aCGH results that may indicate instability and structural mosaicism for ring chromosomes in foetal cells. The presence of a 9 Mb euchromatin region in the analysed sSMC(10) did not lead to clinical manifestations, and a healthy girl was born at term. We suggest that the ring structure of sSMCs could influence sSMC manifestations and should be taken into account in genetic counselling during prenatal diagnosis.

Whole Exome Sequencing Facilitated the Identification of a Mosaic Small Supernumerary Marker Chromosome (sSMC).

Small supernumerary marker chromosomes (sSMCs) are a group of rare chromosomal anomalies, which pose challenges in the clinical practice of prenatal diagnosis and genetic counseling. This study enrolled an extended family with an underage male patient displaying infantile seizures, intellectual disability, and retarded speech and psychomotor function. A series of multiplatform genetic detections was conducted to explore the diagnostic variation. Whole exome sequencing (WES) and chromosomal microarray analysis (CMA) indicated a mosaic sSMC derived from the pericentromeric region of chromosome 8 in the patient, which was confirmed using cytogenetic methods. The proband and his mother, who carried this mosaic variant, exhibited strong phenotypic variability. We also ruled out the pathogenicity of a KDM5C variant by extended validation. Our results emphasized the capacity of WES to detect mosaic SMCs and the importance of mosaic ratios in the appearance and severity of symptomatic phenotypes.

CASC5 Gene Expression Changes Correlate with Targeted Mutations in Leukemia

Dysfunction of genes that control mitosis and are responsible for the correct segregation of sister chromatids in anaphase is often accompanied by aneuploidy, which is frequently detected in leukemia. One of the components of the kinetochore complex, namely, the AF15q14/KNL1/CASC5 protein, is an important factor ensuring the correct binding of the pericentromeric region of chromosomes with the spindle microtubules. As shown recently, in some leukemias, the gene of this protein can be involved in the generation of the chromosomal translocation t(11;15)(q23;q14) or a variant of the chimeric MLL-AF15Q14 oncogene, which serves as a biomarker of poor prognosis. Despite the implication of mRNA of the CASC5 gene in oncogenesis of solid tumors, expression of this gene in hematopoietic neoplasms has not been studied. We analyzed expression levels of the CASC5 gene and the nearest regulatory genes, including WT1, APOBEC3A (A3A), and N-MYC. A pronounced decrease in CASC5 expression in bone marrow cells of primary leukemia patients compared with healthy donors was found. It was also shown that reduced expression of the CASC5 gene correlates with the detection of targeted mutations in patients composed two prognostic subgroups (favorable, unfavorable) with a significance level (p <0.05). It was noted that the change in the expression level of the CASC5 gene in acute myeloid leukemia is associated with overexpression of the genes WT1, A3A, and in some cases N-MYC and SPT16, which is consistent with the resistance to chemotherapy and leukemia progression. However, the question of which regulatory gene initiates leukemogenesis remains open.

Sequence Analysis and FISH Mapping of Four Satellite DNA Families among Cervidae.

Centromeric and pericentromeric chromosome regions are occupied by satellite DNA. Satellite DNAs play essential roles in chromosome segregation, and, thanks to their extensive sequence variability, to some extent, they can also be used as phylogenetic markers. In this paper, we isolated and sequenced satellite DNA I-IV in 11 species of Cervidae. The obtained satellite DNA sequences and their chromosomal distribution were compared among the analysed representatives of cervid subfamilies Cervinae and Capreolinae. Only satI and satII sequences are probably present in all analysed species with high abundance. On the other hand, fluorescence in situ hybridisation (FISH) with satIII and satIV probes showed signals only in a part of the analysed species, indicating interspecies copy number variations. Several indices, including FISH patterns, the high guanine and cytosine (GC) content, and the presence of centromere protein B (CENP-B) binding motif, suggest that the satII DNA may represent the most important satellite DNA family that might be involved in the centromeric function in Cervidae. The absence or low intensity of satellite DNA FISH signals on biarmed chromosomes probably reflects the evolutionary reduction of heterochromatin following the formation of chromosome fusions. The phylogenetic trees constructed on the basis of the satellite I-IV DNA relationships generally support the present cervid taxonomy.

Ski Is Required for Tri-Methylation of H3K9 in Major Satellite and for Repression of Pericentromeric Genes: Mmp3, Mmp10 and Mmp13, in Mouse Fibroblasts

Several mechanisms directing a rapid transcriptional reactivation of genes immediately after mitosis have been described. However, little is known about the maintenance of repressive signals during mitosis. In this work, we address the role of Ski in the repression of gene expression during M/G1 transition in mouse embryonic fibroblasts (MEFs). We found that Ski localises as a distinct pair of dots at the pericentromeric region of mitotic chromosomes, and the absence of the protein is related to high acetylation and low tri-methylation of H3K9 in pericentromeric major satellite. Moreover, differential expression assays in early G1 cells showed that the presence of Ski is significantly associated with repression of genes localised nearby to pericentromeric DNA. In mitotic cells, chromatin immunoprecipitation assays confirmed the association of Ski to major satellite and the promoters of the most repressed genes: Mmp3, Mmp10 and Mmp13. These genes are at pericentromeric region of chromosome 9. In these promoters, the presence of Ski resulted in increased H3K9 tri-methylation levels. This Ski-dependent regulation is also observed during interphase. Consequently, Mmp activity is augmented in Ski−/− MEFs. Altogether, these data indicate that association of Ski with the pericentromeric region of chromosomes during mitosis is required to maintain the silencing bookmarks of underlying chromatin.

Cytogenetic analysis of Bienertia sinuspersici Akhani as the first step in genome sequencing.

C4 plants are efficient in suppressing photorespiration and enhancing carbon gain as compared to C3 plants. Bienertia sinuspersici Akhani is one of the few species in the family Amaranthaceae that can perform C4 photosynthesis within individual chlorenchyma cells, without the conventional Kranz anatomy in its leaf. This plant is salt-tolerant and is well-adapted to thrive in hot and humid climates. To date, there have been no reported cytogenetic analyses yet on this species. This study aims to provide a cytogenetic analysis of B. sinuspersici as the first step in genome sequencing. Fluorescence in situ hybridization (FISH) karyotype analysis was conducted using the metaphase chromosomes of B. sinuspersici probed with 5S and 45S rDNA and Arabidopsis-type telomeric repeats. Results of the cytogenetic analysis confirmed that B. sinuspersici has 2n = 2x = 18 consisting of nine pairs of metacentric chromosomes. Two loci of 45S rDNA were found on the distal regions of the short arm of chromosome 7. Nine loci of 5S rDNA were found in the pericentromeric regions of chromosomes 1, 3, 4, 6, and 8, which also colocalized with Arabidopsis-type telomeric repeats; while four loci in the interstitial regions of chromosome 5 and 8 can be observed. The single locus of 5S rDNA that was found in chromosome 8 appears to be hemizygous. The FISH karyotype analysis, based on the combination of rDNAs, telomeric tandem repeat markers and C0t DNA chromosome landmarks, allowed efficient chromosome identification and provided useful information in characterizing the genome of B. sinuspersici.

The Implication of Cytogenetic Alterations in Pancreatic Ductal Adenocarcinoma and Intraductal Papillary Mucinous Neoplasm Identified by Fluorescence In Situ Hybridization and Their Potential Diagnostic Utility

Background/AimsWe investigated chromosomal aberrations in patients with pancreatic ductal adenocarcinoma (PDAC) and intraductal papillary mucinous neoplasm (IPMN) by fluorescence in situ hybridization (FISH) to identify cytogenetic changes and molecular markers that may be useful for preoperative diagnosis.MethodsTissue samples from 48 PDAC and 17 IPMN patients were investigated by FISH analysis using probes targeting chromosomes 7q, 17p, 18q, 20q, and 21q and the pericentromeric region of chromosome 18 (CEP18).ResultsThe PDAC samples harbored 17p deletion (95.8%), 18q deletion (83.3%), CEP18 deletion (81.2%), 20q gain (81.2%), 21q deletion (77.1%), and 7q gain (70.8%). The IPMN samples had 17p deletion (94.1%), CEP18 deletion (94.1%), 21q deletion (70.6%), 18q deletion (58.8%), 20q gain (58.8%), and 7q gain (58.8%). A significant difference in CEP18 gain was identified between the PDAC and IPMN groups (p=0.029). Detection of 17p or 18q deletion had the highest diagnostic accuracy (80.0%) for PDAC.ConclusionsChromosomal alterations were frequently identified in both PDAC and IPMN with similar patterns. CEP18 gain and 17p and 18q deletions might be involved in the later stages of PDAC tumorigenesis. Chromosome 17p and 18q deletions might be excellent diagnostic markers.

Desiccation-induced changes in recombination rate and crossover interference in Drosophila melanogaster: evidence for fitness-dependent plasticity.

Meiotic recombination is evolutionarily ambiguous, as being associated with both benefits and costs to its bearers, with the resultant dependent on a variety of conditions. While existing theoretical models explain the emergence and maintenance of recombination, some of its essential features remain underexplored. Here we focus on one such feature, recombination plasticity, and test whether recombination response to stress is fitness-dependent. We compare desiccation stress effects on recombination rate and crossover interference in chromosome 3 between desiccation-sensitive and desiccation-tolerant Drosophila lines. We show that relative to desiccation-tolerant genotypes, desiccation-sensitive genotypes exhibit a significant segment-specific increase in single- and double-crossover frequencies across the pericentromeric region of chromosome 3. Significant changes (relaxation) in crossover interference were found for the interval pairs flanking the centromere and extending to the left arm of the chromosome. These results indicate that desiccation is a recombinogenic factor and that desiccation-induced changes in both recombination rate and crossover interference are fitness-dependent, with a tendency of less fitted individuals to produce more variable progeny. Such dependence may play an important role in the regulation of genetic variation in populations experiencing environmental challenges.

Dynamics of the spatial orientation of the pericentromeric heterochromatin regions in the polytene chromosomes of ovarian nurse cells in the Drosophila melanogaster (Diptera: Drosophilidae) oogenesis

The spatial organization of polytene chromosomes and the association of their pericentromeric regions in the Drosophila melanogaster nurse cells during oogenesis have been examined by 3D-immunofluorescence microscopy. All nurse cell chromosomes are shown to contact the nuclear membrane with their pericentromeric regions, and the X chromosome additionally with its telomeric region. Three morphological types of the associations of the nurse cell chromosome pericentromeric regions in the nuclear space are observed: (1) the pericentromeric regions of chromosomes 2, 3, and 4 contact the nuclear membrane at one pole of the nucleus, while the pericentromeric region of the X chromosome does so at the other pole (morphotype I); (2) the pericentromeric regions of chromosomes X, 2, and 3 are separated from each other in the nuclear space and contact the nuclear membrane (morphotype II); and (3) the pericentromeric region of chromosome 2 contacts the nuclear membrane at one pole of the nucleus, while the pericentromeric regions of chromosomes X, 3, and 4 contact the membrane at the other pole (morphotype III). The author, therefore, proposes that the nurse cell nuclei with morphotype I are prevalent at the early stages of oogenesis, while the nurse cell nuclei with morphotype III are most abundant at the late stages. The dynamics of associations of the pericentromeric chromosome regions in the nuclear space of D. melanogaster ovarian nurse cells in the oogenesis demonstrated that there may be some functional relationship between the 3-D organization of the nurse cell chromosomes and the organization of the nucleolus.

Two novel strategies to assess in vivo meiotic protein expression in Arabidopsis thaliana

For studies on key meiotic processes such as crossover formation and genome haploidization, the availability of portable promoter sequences for effector protein expression in meiocytes is of great importance. In this study, we present two novel strategies to facilitate screening for such promoter elements. The first strategy was based on expression of a previously constructed GFP-tagged zinc finger protein for visualization of the pericentromeric regions of chromosomes in meiocytes. Here, we show that expression of this reporter protein under control of different promoters allowed for the visualization of fluorescence foci in meiocytes, demonstrating that this is a useful tool for such purposes. The second reporter system was based on the visualization of cytotoxicity triggered by expression of theAgrobacterium tumefaciensvirulence protein VirD5. We show that constitutive expression of VirD5 is lethal, but when driven by meiotic promoters led to reduced fertility with normal vegetative growth. We show that both strategies offer useful tools for the assessment of meiotic effector protein expression, especially when combined with available gene expression data sets.

Loss of Function of the E1-Like-b Gene Associates With Early Flowering Under Long-Day Conditions in Soybean.

Photoperiod response of flowering determines plant adaptation to different latitudes. Soybean, a short-day plant, has gained the ability to flower under long-day conditions during the growing season at higher latitudes, mainly through dysfunction of phytochrome A genes (E3 and E4) and the floral repressor E1. In this study, we identified a novel molecular genetic basis of photoperiod insensitivity in Far-Eastern Russian soybean cultivars. By testcrossing these cultivars with a Canadian cultivar Harosoy near-isogenic line for a recessive e3 allele, followed by association tests and fine mapping, we determined that the insensitivity was inherited as a single recessive gene located in an 842-kb interval in the pericentromeric region of chromosome 4, where E1-Like b (E1Lb), a homoeolog of E1, is located. Sequencing analysis detected a single-nucleotide deletion in the coding sequence of the gene in insensitive cultivars, which generated a premature stop codon. Near-isogenic lines (NILs) for the loss-of-function allele (designated e1lb) exhibited upregulated expression of soybean FLOWERING LOCUS T (FT) orthologs, FT2a and FT5a, and flowered earlier than those for E1Lb under long-day conditions in both the e3/E4 and E3/E4 genetic backgrounds. These NILs further lacked the inhibitory effect on flowering by far-red light–enriched long-day conditions, which is mediated by E4, but not that of red-light–enriched long-day conditions, which is mediated by E3. These findings suggest that E1Lb retards flowering under long-day conditions by repressing the expression of FT2a and FT5a independently of E1. This loss-of-function allele can be used as a new resource in breeding of photoperiod-insensitive cultivars, and may improve our understanding of the function of the E1 family genes in the photoperiod responses of flowering in soybean.

Single-molecule DNA-mapping and whole-genome sequencing of individual cells

To elucidate cellular diversity and clonal evolution in tissues and tumors, one must resolve genomic heterogeneity in single cells. To this end, we have developed low-cost, mass-producible micro-/nanofluidic chips for DNA extraction from individual cells. These chips have modules that collect genomic DNA for sequencing or map genomic structure directly, on-chip, with denaturation-renaturation (D-R) optical mapping [Marie R, et al. (2013) Proc Natl Acad Sci USA 110:4893-4898]. Processing of single cells from the LS174T colorectal cancer cell line showed that D-R mapping of single molecules can reveal structural variation (SV) in the genome of single cells. In one experiment, we processed 17 fragments covering 19.8 Mb of the cell's genome. One megabase-large fragment aligned well to chromosome 19 with half its length, while the other half showed variable alignment. Paired-end single-cell sequencing supported this finding, revealing a region of complexity and a 50-kb deletion. Sequencing struggled, however, to detect a 20-kb gap that D-R mapping showed clearly in a megabase fragment that otherwise mapped well to the reference at the pericentromeric region of chromosome 4. Pericentromeric regions are complex and show substantial sequence homology between different chromosomes, making mapping of sequence reads ambiguous. Thus, D-R mapping directly, from a single molecule, revealed characteristics of the single-cell genome that were challenging for short-read sequencing.