Longest Chromosome Research Articles

Unveiling the genetic landscape: Exploring the SSR-based genetic architecture and amino acid dissection of Gossypium barbadense and G. darwinii genomes

Genetic maps highlight the genome organization and structure but also provide the chance of tagging superior traits for crop improvement through marker-assisted selection. Amino acids are building blocks of proteins and perform crucial function in regulating the signaling of molecules involved in the development and growth of plants. Plant architecture also have an impact on crop productivity. In order to select elite cultivars for breeding and identification of favorable alleles and their functional properties, a deep understanding of genetic architecture and development of genetic map is essential. In present investigation, an interspecific cross of Gossypium barbadense XH-18 × G. darwinii 5-7 was made to develop a genetic map utilizing single sequence repeat markers for the dissection of amino acids involved in genetic architecture of G. barbadense and G. darwinii. We measured chromosomal distribution of 20 amino acids across the whole genome of both species. The map consists of 613 markers spread across all 26 chromosomes, covering 2371.4 cM of cotton genome with an average inter-marker distance of 9.35 cM. The marker number anchored on the chromosomes varied from 5 to 76 with an average of 23.57 on each chromosome. The Dt sub-genome had more markers (83.03%) than the At sub-genome (15.66%). Moreover, the longest chromosome was 143.387 cM, the shortest was 58.430 cM, and the average length was 91.207 cM. The Dt subgenome spans a greater genomic distance than the At subgenome. A sum of 21,035 genes were discovered, covering the complete genome of G. barbadense; G. darwinii and have been found to be involved in tRNA 3'-trailer cleavage, macromolecule modification, peptide deformylase activity, response to biotic stimulus and defense response. The minimum Glutamic acid (Glu), Histidine (His), and Lysine (Lys) were found on Chr.13 (0.00-17.74), Chr.02 (0.00-8.01), and Chr.06 (0.00-17.97), respectively found through chromosomal amino acid dissection. The genome-wide SSR interspecific genetic map of G. barbadense and G. darwinii is first of its kind, and studying chromosomal distribution of amino acids will set a landmark step to dissect the genome structure of G. darwinii.

Variant in the synaptonemal complex protein SYCE2 associates with pregnancy loss through effect on recombination

Two-thirds of all human conceptions are lost, in most cases before clinical detection. The lack of detailed understanding of the causes of pregnancy losses constrains focused counseling for future pregnancies. We have previously shown that a missense variant in synaptonemal complex central element protein 2 (SYCE2), in a key residue for the assembly of the synaptonemal complex backbone, associates with recombination traits. Here we show that it also increases risk of pregnancy loss in a genome-wide association analysis on 114,761 women with reported pregnancy loss. We further show that the variant associates with more random placement of crossovers and lower recombination rate in longer chromosomes but higher in the shorter ones. These results support the hypothesis that some pregnancy losses are due to failures in recombination. They further demonstrate that variants with a substantial effect on the quality of recombination can be maintained in the population.

Cytogenetical studies of some Convolvulaceae members from the Western Ghats, India reveal uniformity in karyotypes

Karyotypes of six Ipomoea and one Merremia species were studied. Chromosome counts of 2n = 30 for I. corymbosa, I. kotschyana, I. marginata f. candida and M. rhyncorhiza) and 2n = 60 for I. ochracea were observed for the first time. Meiotic course of five Ipomoea species was also examined for the first time and two counts, i.e. n = 15 and n = 30 were recorded. Meiosis was found normal. Karyotypes of the studied species exhibited uniformity. All the species had chromosomes with median region centromeres and karyotypes were symmetrical (Stebbins’ 4A category). I. marginata f. candida had the shortest chromosomes with mean length of 0.99 ± 0.02 µm. I. ochracea and I. corymbosa had the longest chromosomes with mean length of 1.22 ± 0.05 µm and 1.22 ± 0.01 µm, respectively. As chromosomes were small and exhibited uniform morphology, fluorescent banding or fluorescent in-situ hybridization can be useful to differentiate the karyotypes and understand species interrelationships.

Unraveling Large and Polyploidy Genome of the Crucifer Orychophragmus violaceus in China, a Potential Oil Crop.

The genus Orychophragmus in the Brassicaceae family includes the types with 2n = 20, 22, 24, and 48. The species O. violaceus (L.) O. E. Schulz has 2n = 24 and is widely cultivated as an ornamental plant in China. This review summarizes the research progress of its genome structure and evolution in the context of cytogenetics and genome sequencing. This species has a large genome size of ~1 Gb and longer chromosomes than those of Brassica species, which is attributable to the burst of TE insertions. Even more, one tetraploidization event from about 600-800 million years ago is elucidated to occur during its genome evolution, which is consistent with the polyploidy nature of its genome revealed by the meiotic pairing patterns. Its chromosomes are still characterized by a larger size and deeper staining than those from Brassica species in their intergeneric hybrids, which is likely related to their inherent differences between genome structures and cytology. Its genome is dissected by the development of additional alien lines, and some traits are located on individual chromosomes. Due to the abundant dihydroxy fatty acids in its seed oil with superior lubricant properties and wide environmental adaptations, this plant promises to be utilized as one new oil crop in the future.

Fuzzy clustering decomposition of genetic algorithm-based instance selection for regression problems

Data selection, which includes feature and instance selection, is often an important step in building prediction systems. Genetic algorithms (GA) frequently allow finding better solutions than classical methods in many areas. This is also true for the instance selection task. The main difficulties and challenges in GA-based instance selection are high computational complexity and decreasing performance with the dataset size growth. This is caused by the fact that each instance is encoded in one chromosome position. Hence bigger datasets result in longer chromosomes. The main contribution of this paper addresses the above problems in a three-step process. In the first step the dataset is divided into several consistent regions by fuzzy clustering. Then GA-based instance selection is performed independently within each cluster. Finally ensemble voting provides seamless aggregation of the partial results from the overlapping clusters. This improves dataset exploitation by more localized search and also takes the advantage of ensemble methods. This method significantly improves the predictive model performance and data reduction in comparison to instance selection performed on the whole training dataset.

The draft chromosome-level genome assembly of tetraploid ground cherry (Prunus fruticosa Pall.) from long reads

Cherries are stone fruits and belong to the economically important plant family of Rosaceae with worldwide cultivation of different species. The ground cherry, Prunus fruticosa Pall., is an ancestor of cultivated sour cherry, an important tetraploid cherry species. Here, we present a long read chromosome-level draft genome assembly and related plastid sequences using the Oxford Nanopore Technology PromethION platform and R10.3 pore type. We generated a final consensus genome sequence of 366 Mb comprising eight chromosomes. The N50 scaffold was ~44 Mb with the longest chromosome being 66.5 Mb. The chloroplast and mitochondrial genomes were 158,217 bp and 383,281 bp long, which is in accordance with previously published plastid sequences. This is the first report of the genome of ground cherry (P. fruticosa) sequenced by long read technology only. The datasets obtained from this study provide a foundation for future breeding, molecular and evolutionary analysis in Prunus studies.

Cytogenetic markers using single-sequence probes reveal chromosomal locations of tandemly repetitive genes in scleractinian coral Acropora pruinosa

The short and similar sized chromosomes of Acropora pose a challenge for karyotyping. Conventional methods, such as staining of heterochromatic regions, provide unclear banding patterns that hamper identification of such chromosomes. In this study, we used short single-sequence probes from tandemly repetitive 5S ribosomal RNA (rRNA) and core histone coding sequences to identify specific chromosomes of Acropora pruinosa. Both the probes produced intense signals in fluorescence in situ hybridization, which distinguished chromosome pairs. The locus of the 5S rDNA probe was on chromosome 5, whereas that of core histone probe was on chromosome 8. The sequence of the 5S rDNA probe was composed largely of U1 and U2 spliceosomal small nuclear RNA (snRNA) genes and their interspacers, flanked by short sequences of the 5S rDNA. This is the first report of a tandemly repetitive linkage of snRNA and 5S rDNA sequences in Cnidaria. Based on the constructed tentative karyogram and whole genome hybridization, the longest chromosome pair (chromosome 1) was heteromorphic. The probes also hybridized effectively with chromosomes of other Acropora species and population, revealing an additional core histone gene locus. We demonstrated the applicability of short-sequence probes as chromosomal markers with potential for use across populations and species of Acropora.

Analisis Kromosom Varietas Kacang Gude (Cajanus cajan (L.) Millspaugh) Asal Pulau Kisar, Maluku Barat Daya (MBD)

Pigeon pea (Cajanus cajan) is one of the biodiversity of Kisar Island, Maluku Barat Daya (MBD). This study aimed to determine the number and size of chromosomes from pigeon pea (C. cajan) from Kisar Island, MBD. The method used for chromosome observation is the squashing method, while for the analysis of the shape and size of chromosomes using the picsart and image raster applications. Data analysis using descriptive analysis with the help of figures and tables. The results showed that the chromosome number of three varieties of pigeon pea (C. cajan) was 2n = 14. The longest chromosome is owned by orange seed varieties that are 9.17 µm, batik color seed varieties are 9.09 μm, the shortest chromosomes are owned by black seed varieties of 8.32 µm. The chromosome length of the three varieties of pigeon pea (C. cajan) varies showing different genome numbers.   Key words: Chromosome; Cajanus cajan; Kisar Island; Maluku Barat Daya. Â

Karyotype analysis and chromosome number for two Cirsium taxa (Asteraceae) in Iran

Cirsium Mill. contains more than 250 species in the world mainly distributed in the Northern hemisphere. Different chromosome numbers with different ploidy levels were reported in this genus. In this study, karyotype details and chromosome numbers were established for two Cirsium taxa in Iran. C. ciliatum subsp. szovitsii and C. echinus had the mitotic chromosome numbers of 2n = 2x = 34. Karyotype analyses showed that chromosomes were generally metacentric and sub-metacentric. In C. echinus, Lowshan population had the longest chromosome (19.10 µm) and Heyran Canyon population (4.73 µm) the shortest one while in C. ciliatum, the longest chromosome was observed in Urmia to Salmas population (14.67 µm) and the shortest one (4.71 µm) in Doshanlu population. Total haploid chromosome length ranged from 275.29 to 376.42 µm in populations studied. Both taxa were grouped in 2B class. B-chromosomes were recorded for two taxa studied too. Chromosome type, mitotic chromosome numbers and occurrence of B-chromosomes were in agreement with previous results (Albers, Pröbsting, 1998; Lövkvist, Hultgård, 1999; Yüksel et al., 2013; Yildiz et al., 2016).

The Incidence of Mosaicism for Individual Chromosome in Human Blastocysts Is Correlated With Chromosome Length.

Mosaicism, known as partial aneuploidies, mostly originates from mitotic errors during the post-zygotic stage; it consists of different cell lineages within a human embryo. The incidence of mosaicism has not been shown to correlate with maternal age, and its correlation with individual chromosome characteristics has not been well investigated. In this study, the results of preimplantation genetic testing for aneuploidy (PGT-A) derived from 4,036 blastocysts (930 IVF couples) were collected from 2015 to 2017. Via next-generation sequencing for comprehensive chromosome screening, embryo ploidy was identified as aneuploid, mosaic, and euploid. Total mosaicism was classified into two categories: “mosaic euploid/aneuploidy” (with mosaic aneuploidy between 20 and 80%) and “mosaic and aneuploidy” (a uniformly abnormal embryo superimposed with mosaic aneuploidies). Frequency of mosaicism was analyzed according to the function of chromosomal lengths, which divides involved chromosomes into three groups: group A (156–249 Mb), group B (102–145 Mb), and group C (51–90 Mb). The results show that the aneuploidy was more frequent in group C than in group A and group B (A: 23.7%, B: 35.1, 41.2%, p < 0.0001), while the mosaicism was more frequent in group A and group B than in group C [(Mosaic euploid/aneuploid) A: 14.6%, B: 12.4%, C: 9.9%, p < 0.0001; (mosaic and aneuploid) A: 21.3%, B: 22.9%, C: 18.9%, p < 0.0001; (Total mosaicism) A: 35.9%, B: 35.3%, C: 28.8%, p < 0.0001]. The significantly higher frequency of aneuploidy was on the shorter chromosome (< 90 Mb), and that of mosaicism was on the longer chromosomes (> 100 Mb). The length association did not reach significance in the patients with advanced age (≥ 36 years), and of the chromosome-specific mosaicism rate, the highest prevalence was on chromosome 14 (5.8%), 1 (5.7%), and 9 (5.6%). Although the length association was observed via group comparison, there may be affecting mechanisms other than chromosomes length. Eventually, twenty patients with mosaic embryo cryotransfers resulted in six live births. No significant correlation was observed between the transfer outcomes and chromosome length; however, the analysis was limited by small sample size.

Micromorphological and cytological comparisons between Youngia japonica and Youngia longiflora using light and scanning electron microscopy.

The genus Youngia Cass. is taxonomically complex and widely distributed in East Asia. Despite the high diversity of species, only two species, Youngia japonica (L.) DC. and Youngia longiflora (Babc. & Stebbins) C.Shih, are currently found in Korea. Among them, Y. longiflora was once recognized as a subspecies of Y. japonica. However, based on chloroplast and nuclear DNA sequences, as well as morphology, Y. longiflora was found to be a distinct species. Here, the two Korean Youngia species were analyzed using scanning electron microscope and light microscope for chromosome number, pollen, cypselae, and leaf micromorphological characters to evaluate if the examined characters are informative for taxonomy. Pollen morphology is described, and the micromorphological characters of Y. longiflora are described for the first time. Cypselae and pollen surface ornamentation, as well as trichome morphology and stomatal size varied between the two species. Y. japonica had longer chromosomes than Y. longiflora, while other chromosomal characters including the localization of the NOR (nucleolar organizer region) were similar. The present micromorphological and cytological results are of great taxonomic value for Youngia species identification in Korea.

Triploidy in Chinese parthenogenetic Helophorus orientalis Motschulsky, 1860, further data on parthenogenetic H. brevipalpis Bedel, 1881 and a brief discussion of parthenogenesis in Hydrophiloidea (Coleoptera).

The chromosomes of triploid parthenogenetic Helophorus orientalis Motschulsky, 1860 are described from material from two localities in Heilongjiang, China. 3n = 33. All the chromosomes have clear centromeric C-bands, and in the longest chromosome one replicate appears to be consistently longer than the other two. The chromosomes of additional triploid parthenogenetic H. brevipalpis Bedel, 1881, from Spain and Italy, are described. In one Italian population one of the autosomes is represented by only two replicates and another appears more evenly metacentric than in material from Spain and the other Italian locality. Parthenogenetic and bisexual specimens of H. orientalis are illustrated, along with Pleistocene fossil material. Parthenogenetic H. brevipalpis is also illustrated. Parthenogenesis in Hydrophiloidea is discussed. It appears to be rare and, in all cases has been detected by chromosomal analysis of populations in which males are unexpectedly scarce. Parthenogenesis is suspected in Helophorus aquila Angus et al., 2014, from northern Qinghai (China), which should be verified in further studies.

Genome-wide identification and transcriptome profiling reveal that E3 ubiquitin ligase genes relevant to ethylene, auxin and abscisic acid are differentially expressed in the fruits of melting flesh and stony hard peach varieties

BackgroundUbiquitin ligases (E3) are the enzymes in the ubiquitin/26S proteasome pathway responsible for targeting proteins to the degradation pathway and play major roles in multiple biological activities. However, the E3 family and their functions are yet to be identified in the fruit of peach.ResultsIn this study, genome-wide identification, classification and characterization of the E3 ligase genes within the genome of peach (Prunus persica) was carried out. In total, 765 E3 (PpE3) ligase genes were identified in the peach genome. The PpE3 ligase genes were divided into eight subfamilies according to the presence of known functional domains. The RBX subfamily was not detected in peach. The PpE3 ligase genes were not randomly distributed among the 8 chromosomes, with a greater concentration on the longer chromosomes. The primary mode of gene duplication of the PpE3 ligase genes was dispersed gene duplication (DSD). Four subgroups of the BTB subfamily never characterized before were newly identified in peach, namely BTBAND, BTBBL, BTBP and BTBAN. The expression patterns of the identified E3 ligase genes in two peach varieties that display different types of fruit softening (melting flesh, MF, and stony hard, SH) were analyzed at 4 different stages of ripening using Illumina technology. Among the 765 PpE3 ligase genes, 515 (67.3%) were expressed (FPKM > 1) in the fruit of either MF or SH during fruit ripening. In same-stage comparisons, 231 differentially expressed genes (DEGs) were identified between the two peach cultivars. The number of DEGs in each subfamily varied. Most DEGs were members of the BTB, F-box, U-box and RING subfamilies. PpE3 ligase genes predicted to be involved in ethylene, auxin, or ABA synthesis or signaling and DNA methylation were differentially regulated. Eight PpE3 ligase genes with possible roles in peach flesh texture and fruit ripening were discussed.ConclusionsThe results of this study provide useful information for further understanding the functional roles of the ubiquitin ligase genes in peach. The findings also provide the first clues that E3 ligase genes may function in the regulation of peach ripening.

Revisiting the Phylogenetic History of Helminths Through Genomics, the Case of the New Echinococcus oligarthrus Genome.

The first parasitic helminth genome sequence was published in 2007; since then, only ∼200 genomes have become available, most of them being draft assemblies. Nevertheless, despite the medical and economical global impact of helminthic infections, parasite genomes in public databases are underrepresented. Recently, through an integrative approach involving morphological, genetic, and ecological aspects, we have demonstrated that the complete life cycle of Echinococcus oligarthrus (Cestoda: Taeniidae) is present in South America. The neotropical E. oligarthrus parasite is capable of developing in any felid species and producing human infections. Neotropical echinococcosis is poorly understood yet and requires a complex medical examination to provide the appropriate intervention. Only a few cases of echinococcosis have been unequivocally identified and reported as a consequence of E. oligarthrus infections. Regarding phylogenetics, the analyses of mitogenomes and nuclear datasets have resulted in discordant topologies, and there is no unequivocal taxonomic classification of Echinococcus species so far. In this work, we sequenced and assembled the genome of E. oligarthrus that was isolated from agoutis (Dasyprocta azarae) naturally infected and performed the first comparative genomic study of a neotropical Echinococcus species. The E. oligarthrus genome assembly consisted of 86.22 Mb which showed ∼90% identity and 76.3% coverage with Echinococcus multilocularis and contained the 85.0% of the total expected genes. Genetic variants analysis of whole genome revealed a higher rate of intraspecific genetic variability (23,301 SNPs; 0.22 SNPs/kb) rather than for the genomes of E. multilocularis and Echinococcus canadensis G7 but lower with respect to Echinococcus granulosus G1. Comparative genomics against E. multilocularis, E. granulosus G1, and E. canadensis G7 revealed 38,762, 125,147, and 170,049 homozygous polymorphic sites, respectively, indicating a higher genetic distance between E. oligarthrus and E. granulosus sensu lato species. The SNP distribution in chromosomes revealed a higher SNP density in the longest chromosomes. Phylogenetic analysis using whole-genome SNPs demonstrated that E. oligarthrus is one of the basal species of the genus Echinococcus and is phylogenetically closer to E. multilocularis. This work sheds light on the Echinococcus phylogeny and settles the basis to study sylvatic Echinococcus species and their developmental evolutionary features.

Karyological and Chromosome Analysis of Quercus libani in Iran

This study attempts to analyze the karyotype and chromosome structure in five populations of Quercus libani growing in Northern Zagros. Pre-treatment, fixation, hydrolysis and staining were conducted using root meristem and then microscopic samples were prepared and studied. The results showed that in all studied cells of each population, the basic chromosome number was x=12 and all of them were diploid. Karyotype analysis of each population was conducted separately and several indices (TL: Total Length, LA: Long Arm, SA: Short Arm, CI: Centromer Index, AR: Arm Ratio, R- value, DRL%: Difference of Relative Length and TF%: Total Form) were determined. Karyotype formula was 12m in all studied populations. The length of chromosomes in all populations was estimated as 0.64-2.08 micrometers. The longest chromosome was observed in chromosome number 1 from population 1 and the shortest one was related to the chromosome number 12 from population 5. Considering of chromosomal classification, all the studied populations were placed in class B of Stebbins which showed that there is an average symmetry in the studied karyotypes. The other estimated indices also showed that in chromosomes are relatively symmetric in all populations that indicates this species is primitive and undeveloped.

HO Endonuclease-Initiated Recombination in Yeast Meiosis Fails To Promote Homologous Centromere Pairing and Is Not Constrained To Utilize the Dmc1 Recombinase.

Crossover recombination during meiosis is accompanied by a dramatic chromosome reorganization. In Saccharomyces cerevisiae, the onset of meiotic recombination by the Spo11 transesterase leads to stable pairwise associations between previously unassociated homologous centromeres followed by the intimate alignment of homologous axes via synaptonemal complex (SC) assembly. However, the molecular relationship between recombination and global meiotic chromosome reorganization remains poorly understood. In budding yeast, one question is why SC assembly initiates earliest at centromere regions while the DNA double strand breaks (DSBs) that initiate recombination occur genome-wide. We targeted the site-specific HO endonuclease to various positions on S. cerevisiae’s longest chromosome in order to ask whether a meiotic DSB’s proximity to the centromere influences its capacity to promote homologous centromere pairing and SC assembly. We show that repair of an HO-mediated DSB does not promote homologous centromere pairing nor any extent of SC assembly in spo11 meiotic nuclei, regardless of its proximity to the centromere. DSBs induced en masse by phleomycin exposure likewise do not promote homologous centromere pairing nor robust SC assembly. Interestingly, in contrast to Spo11, HO-initiated interhomolog recombination is not affected by loss of the meiotic kinase, Mek1, and is not constrained to use the meiosis-specific Dmc1 recombinase. These results strengthen the previously proposed idea that (at least some) Spo11 DSBs may be specialized in activating mechanisms that both 1) reinforce homologous chromosome alignment via homologous centromere pairing and SC assembly, and 2) establish Dmc1 as the primary strand exchange enzyme.

Genome sequence comparison between Chinese hamster ovary (CHO) DG44 cells and mouse using end sequences of CHO BAC clones based on BAC-FISH results.

Chinese hamster ovary (CHO) cells have frequently been used in biotechnology as a mammalian host cell platform for expressing genes of interest. Previously, we constructed a detailed physical chromosomal map of the CHO DG44 cell line by fluorescence in situ hybridization (FISH) imaging using 303 bacterial artificial chromosome (BAC) clones as hybridization probes (BAC-FISH). BAC-FISH results revealed that the two longest chromosomes were completely paired. However, other chromosomes featured partial deletions or rearrangements. In this study, we determined the end sequences of 303 BAC clones (BAC end sequences), which were used for BAC-FISH probes. Among 606 BAC-end sequences (BESs) (forward and reverse ends), 558 could be determined. We performed a comparison between all determined BESs and mouse genome sequences using NCBI BLAST. Among these 558 BESs, 465 showed high homology to mouse chromosomal sequences. We analyzed the locations of these BACs in chromosomes of the CHO DG44 cell line using a physical chromosomal map. From the obtained results, we investigated the regional similarities among CHO chromosomes (A-T) and mouse chromosomes (1-19 and sex) about 217 BESs (46.7% of 465 high homologous BESs). Twenty-three specific narrow regions in 13 chromosomes of the CHO DG44 cell line showed high homology to mouse chromosomes, but most of other regions did not show significant correlations with the mouse genome. These results contribute to accurate alignments of chromosomes of Chinese hamster and its genome sequence, analysis of chromosomal instability in CHO cells, and the development of target locations for gene and/or genome editing techniques.

Multiple tolerance defects contribute to the breach of B cell tolerance in New Zealand Black chromosome 1 congenic mice.

Lupus is characterized by a loss of B cell tolerance leading to autoantibody production. In this study, we explored the mechanisms underlying this loss of tolerance using B6 congenic mice with an interval from New Zealand Black chromosome 1 (denoted c1(96–100)) sufficient for anti-nuclear antibody production. Transgenes for soluble hen egg white lysozyme (sHEL) and anti-HEL immunoglobulin were crossed onto this background and various tolerance mechanisms examined. We found that c1(96–100) mice produced increased levels of IgM and IgG anti-HEL antibodies compared to B6 mice and had higher proportions of germinal center B cells and long-lived plasma cells, suggesting a germinal center-dependent breach of B cell anergy. Consistent with impaired anergy induction, c1(96–100) double transgenic B cells showed enhanced survival and CD86 upregulation. Hematopoietic chimeric sHEL mice with a mixture of B6 and c1(96–100) HEL transgenic B cells recapitulated these results, suggesting the presence of a B cell autonomous defect. Surprisingly, however, there was equivalent recruitment of B6 and c1(96–100) B cells into germinal centers and differentiation to splenic plasmablasts in these mice. In contrast, there were increased proportions of c1(96–100) T follicular helper cells and long-lived plasma cells as compared to their B6 counterparts, suggesting that both B and T cell defects are required to breach germinal center tolerance in this model. This possibility was further supported by experiments showing an enhanced breach of anergy in double transgenic mice with a longer chromosome 1 interval with additional T cell defects.

Karyomorphology and nuclear DNA content of sixteen Ophrys L. taxa from Turkey

Karyotypes in 16 representative taxa of the Ophrys genus are compared, based on Feulgen-stained somatic metaphase chromosomes. The karyotypes of O. omegaifera subsp. israelitica, O. ulupinara, O. lycia, O. argolica subsp. lucis, O. argolica subsp. lesbis, O. climacis and O. reinholdii subsp. reinholdii are described for the first time. Karyological analyses indicate relationships among the species with respect to their asymmetry indices. All Ophrys taxa studied were diploid with 2n = 2x = 36 chromosomes. One B chromosome has been detected among the chromosomes of O. argolica subsp. lucis. All karyotypes are symmetrical, consisting of metacentric and submetacentric chromosomes. The longest chromosomes of all the investigated specimens contain a secondary constriction. It is determined that there is a correlation between the total number of chromosomes having secondary constrictions and the evolutionary development order of the taxa. Based on nuclear DNA content, analysis was carried out by flow cytometer using propodium iodide as fluorochrome, 2C nuclear DNA content of 16 Ophrys species varying between 20.80 pg (O. argolica subsp. lucis) and 23.11 pg (O. omegaifera subsp. israelitica). Karyotype asymmetry relationships are discussed according to the bidimensional scatter plots of A1–A2, CVCL–CVCI, CVCL–MCA and CVCI–MCA.

Characterization of the conbercept gene localization in DHFR- amplified CHO cells using fluorescence in situ hybridization.

Chinese-hamster ovary (CHO) cells are most widely used for mammalian protein expression. After integration into the CHO genome, the exogenous gene may be lost in the process of large-scale protein production due to the removal of related selection pressures. Therefore, it is necessary to test its stability in the genome. Conbercept is a fusion protein that specifically binds to the various isoforms of vascular endothelial growth factor (VEGF)-A, VEGF-B, and placental growth factor (PlGF), thereby exerting anti-angiogenic activities. It has been approved for Phase Ⅲ clinical trials in the United States. In this study, fluorescence in situ hybridization (FISH) was used to localize the conbercept gene in dihydrofolatereductase (DHFR)-amplified CHO cell lines. Metaphase FISH showed that genomic integration of the conbercept gene was stable after 4 and 19 passages, and manifested three characteristics: first, the gene locates on one chromosome, rather than a number of chromosomes; second, the gene locates on the longer chromosomes; third, there are many copies located on the same chromosome. At the same time, the copy number of the conbercept gene in the CHO genome and the conbercept protein expression levels are also stable, as verified by qPCR and ELISA assays, respectively. These experiments demonstrated that the conbercept gene remained stable in the genome after 19 passages, and could be actively expressed, which strongly support the mass production and the quality control of conbercept.