Single Diploid Cell Research Articles

Diploid Nuclei Occur throughout the Life Cycles of Pucciniales Fungi.

Within Eukaryotes, fungi are the typical representatives of haplontic life cycles. Basidiomycota fungi are dikaryotic in extensive parts of their life cycle, but diploid nuclei are known to form only in basidia. Among Basidiomycota, the Pucciniales are notorious for presenting the most complex life cycles, with high host specialization, and for their expanded genomes. Using cytogenomic (flow cytometry and cell sorting on propidium iodide-stained nuclei) and cytogenetic (FISH with rDNA probe) approaches, we report the widespread occurrence of replicating haploid and diploid nuclei (i.e., 1C, 2C and a small proportion of 4C nuclei) in diverse life cycle stages (pycnial, aecial, uredinial, and telial) of all 35 Pucciniales species analyzed, but not in sister taxa. These results suggest that the Pucciniales life cycle is distinct from any cycle known, i.e., neither haplontic, diplontic nor haplodiplontic, corroborating patchy and disregarded previous evidence. However, the biological basis and significance of this phenomenon remain undisclosed. IMPORTANCE Within Eukaryotes, fungi are the typical representatives of haplontic life cycles, contrasting with plants and animals. As such, fungi thus contain haploid nuclei throughout their life cycles, with sexual reproduction generating a single diploid cell upon karyogamy that immediately undergoes meiosis, thus resuming the haploid cycle. In this work, using cytogenetic and cytogenomic tools, we demonstrate that a vast group of fungi presents diploid nuclei throughout their life cycles, along with haploid nuclei, and that both types of nuclei replicate. Moreover, haploid nuclei are absent from urediniospores. The phenomenon appears to be transversal to the organisms in the order Pucciniales (rust fungi) and it does not occur in neighboring taxa, but a biological explanation or function for it remains elusive.

Imputation-free reconstructions of three-dimensional chromosome architectures in human diploid single-cells using allele-specified contacts

Single-cell Hi-C analysis of diploid human cells is difficult because of the lack of dense chromosome contact information and the presence of homologous chromosomes with very similar nucleotide sequences. Thus here, we propose a new algorithm to reconstruct the three-dimensional (3D) chromosomal architectures from the Hi-C dataset of single diploid human cells using allele-specific single-nucleotide variations (SNVs). We modified our recurrence plot-based algorithm, which is suitable for the estimation of the 3D chromosome structure from sparse Hi-C datasets, by newly incorporating a function of discriminating SNVs specific to each homologous chromosome. Here, we eventually regard a contact map as a recurrence plot. Importantly, the proposed method does not require any imputation for ambiguous segment information, but could efficiently reconstruct 3D chromosomal structures in single human diploid cells at a 1-Mb resolution. Datasets of segments without allele-specific SNVs, which were considered to be of little value, can also be used to validate the estimated chromosome structure. Introducing an additional mathematical measure called a refinement further improved the resolution to 40-kb or 100-kb. The reconstruction data supported the notion that human chromosomes form chromosomal territories and take fractal structures where the dimension for the underlying chromosome structure is a non-integer value.

Mammalian Spermatozoa: Physiological, Biochemical and Biotechnological Aspects

Fertilization, an important phase of reproduction, is physiologically the union of the male and female gamete. This succession involves the fusion of an oocyte with a sperm, leading to generation of a single diploid cell, the zygote, from which a new individual organism develops. The elucidation of the physiological, biochemical and molecular mechanisms of fertilization has enthralled researchers of relevance for several years. This editorial embodies this fascinating succession at physiological, biochemical and molecular level.

Sharing Marks: H3K4 Methylation and H2B Ubiquitination as Features of Meiotic Recombination and Transcription.

Meiosis is a specialized cell division that gives raise to four haploid gametes from a single diploid cell. During meiosis, homologous recombination is crucial to ensure genetic diversity and guarantee accurate chromosome segregation. Both the formation of programmed meiotic DNA double-strand breaks (DSBs) and their repair using homologous chromosomes are essential and highly regulated pathways. Similar to other processes that take place in the context of chromatin, histone posttranslational modifications (PTMs) constitute one of the major mechanisms to regulate meiotic recombination. In this review, we focus on specific PTMs occurring in histone tails as driving forces of different molecular events, including meiotic recombination and transcription. In particular, we concentrate on the influence of H3K4me3, H2BK123ub, and their corresponding molecular machineries that write, read, and erase these histone marks. The Spp1 subunit within the Complex of Proteins Associated with Set1 (COMPASS) is a critical regulator of H3K4me3-dependent meiotic DSB formation. On the other hand, the PAF1c (RNA polymerase II associated factor 1 complex) drives the ubiquitination of H2BK123 by Rad6-Bre1. We also discuss emerging evidence obtained by cryo-electron microscopy (EM) structure determination that has provided new insights into how the “cross-talk” between these two marks is accomplished.

New strategies in the field of mixture deconvolution single cell STR profiling

STR profiling of single cells isolated by DEPArray™ technology can be used for the successful deconvolution of mixtures composed of cells of the same cell type. In order to gain more information about characteristics and quality of profiles derived from single diploid cells that were separated with the technology mentioned above, a validation study was carried out. Application-specific interpretation guidelines were determined. Under the aspect of occurring drop ins it’s essential to always combine several partial profiles attributed to the same person to a consensus profile in order to ensure the derivation of the correct profile. Taking this approach into account, the technique opens up a completely new dimension of mixed stain processing. As shown by an example, this, in principle, also applies to single sperm (haploid cells).

Diploid genome in 3D.

Dip-C, a descendent of the 3C method, reveals 3D genome structures of single diploid human cells.

Three-dimensional genome structures of single diploid human cells.

Three-dimensional genome structures play a key role in gene regulation and cell functions. Characterization of genome structures necessitates single-cell measurements. This has been achieved for haploid cells but has remained a challenge for diploid cells. We developed a single-cell chromatin conformation capture method, termed Dip-C, that combines a transposon-based whole-genome amplification method to detect many chromatin contacts, called META (multiplex end-tagging amplification), and an algorithm to impute the two chromosome haplotypes linked by each contact. We reconstructed the genome structures of single diploid human cells from a lymphoblastoid cell line and from primary blood cells with high spatial resolution, locating specific single-nucleotide and copy number variations in the nucleus. The two alleles of imprinted loci and the two X chromosomes were structurally different. Cells of different types displayed statistically distinct genome structures. Such structural cell typing is crucial for understanding cell functions.

The structure of the genome

Genome Structure Beyond the sequence of the genome, its three-dimensional structure is important in regulating gene expression. To understand cell-to-cell variation, the structure needs to be understood at a single-cell level. Chromatin conformation capture methods have allowed characterization of genome structure in haploid cells. Now, Tan et al. report a method called Dip-C that allows them to reconstruct the genome structures of single diploid human cells. Their examination of different cell types highlights the tissue dependence of three-dimensional genome structures. Science , this issue p. [924][1] [1]: /lookup/doi/10.1126/science.aat5641

Poczęcie człowieka i status jednokomórkowej ludzkiej zygoty z perspektywy bioetyki katolickiej

In the discussions on the status of human zygote, the issue of its animation appears only occasionally. Current teaching of the Magisterium of the Church on the beginning of human life emphasises the fact that life of a new human being begins with the conception. The moment when single 2n diploid cell called zygote comes into being is very often considered as a start of a new human life. According to embryology, this takes place 12 to 24 hours after fertilization. The discoveries in epigenetics allow to claim the human character of a zygote is then defined. From the moment of conception, that is when the head of spermatozoon combines with oocyte cytoplasm and two pronuclei come into existence (the nuclei of both male and female gametes), the processes related to a new human life begin.

The molecular basis of fertilization (Review).

Fertilization is the fusion of the male and female gamete. The process involves the fusion of an oocyte with a sperm, creating a single diploid cell, the zygote, from which a new individual organism will develop. The elucidation of the molecular mechanisms of fertilization has fascinated researchers for many years. In this review, we focus on this intriguing process at the molecular level. Several molecules have been identified to play a key role in each step of this intriguing process (the sperm attraction from the oocyte, the sperm maturation, the sperm and oocyte fusion and the two gamete pronuclei fusion leading to the zygote). Understanding the molecular mechanisms of the cell-cell interactions will provide a better understanding of the causes of fertility issues due to fertilization defects.

Natural variation in epigenetic pathways affects the specification of female gamete precursors in Arabidopsis.

In angiosperms, the transition to the female gametophytic phase relies on the specification of premeiotic gamete precursors from sporophytic cells in the ovule. In Arabidopsis thaliana, a single diploid cell is specified as the premeiotic female gamete precursor. Here, we show that ecotypes of Arabidopsis exhibit differences in megasporogenesis leading to phenotypes reminiscent of defects in dominant mutations that epigenetically affect the specification of female gamete precursors. Intraspecific hybridization and polyploidy exacerbate these defects, which segregate quantitatively in F2 populations derived from ecotypic hybrids, suggesting that multiple loci control cell specification at the onset of female meiosis. This variation in cell differentiation is influenced by the activity of ARGONAUTE9 (AGO9) and RNA-DEPENDENT RNA POLYMERASE6 (RDR6), two genes involved in epigenetic silencing that control the specification of female gamete precursors. The pattern of transcriptional regulation and localization of AGO9 varies among ecotypes, and abnormal gamete precursors in ovules defective for RDR6 share identity with ectopic gamete precursors found in selected ecotypes. Our results indicate that differences in the epigenetic control of cell specification lead to natural phenotypic variation during megasporogenesis. We propose that this mechanism could be implicated in the emergence and evolution of the reproductive alternatives that prevail in flowering plants.

316 GENOMIC EVALUATION OF BOVINE EMBRYOS WITHIN 24 HOURS

The aim of this study was to develop a reliable procedure for genomic evaluation of bovine embryos to determine gender, polled status, and hereditary defects within 24 h after collection. German Simmental animals (n = 15) were superovulated (n = 25) using a standard protocol. Embryos were recovered on Day 7 (Day 0 = oestrus). A total of 217 embryos (morula, n = 130; early blastocyst, n = 43; blastocyst, n = 44) were biopsied with a steel blade attached to a micromanipulator. Biopsied cells were immediately transferred into 1 µL TE buffer to a 500 µL reaction tube and embryos were in vitro cultured until genomic results were available. For commonly used molecular genetic methods (e.g. 5′-exonuclease genotyping, PCR or high density genotyping) DNA amounts of 2–200 ng are required. However, the DNA quantity of a single diploid cell amounts to 6 pg only. The embryo biopsies used, usually consists of 10–30 cells, necessitating an artificial amplification of the embryonic genome. Taking all vital measures to avoid external DNA contamination, isothermal whole genome amplification was performed with the REPLI-g Mini Kit (Qiagen, Valencia, CA, USA) using random hexamers and Phi29-Polymerase. Depending on the number of cells, a total DNA amount of 4–7 µg was achieved. Polled status and gender was determined using PCR with subsequent gel-electrophoresis. 5′-exonuclease assays were used to obtain genotypes for the detection of genetic defects. At present, eight, mostly Simmental-specific genetic disorders can be examined: three traits associated with severe growth retardation, dwarfism (DW), Braunvieh-haplotype 2 (BH2) and stunted growth (FH2), the lethal skin disorder zinc deficiency-like syndrome (ZDL), a fertility trait bovine male subfertility (BMS), embryonic death Fleckvieh-haplotype 4 (FH4), a bleeding disorder thrombopathia (TP) and arachnomelia (A), within 24 h. On average, 8.7 embryos were biopsied per embryo recovery, i.e. 93.9% of the total number of transferable embryos. Fourteen embryo samples (6.5%) totally failed during analysis, possibly due to the loss of samples. In successful analyses, gender was undetermined in two embryos; remaining embryos were 52.2% female and 47.8% male. Polled status could be analysed in 92.6% of the embryos. The analyses of embryos for possible inherited genetic disorders (healthy, heterozygote, or homozygote; n = 578) were successful in 90.1%. The transfer of biopsied embryos (n = 30) led to 63.3% pregnancies (Day 42). A validation of the present results has to be done as soon as the produced calves are born, demonstrating the reliability of the procedure.Research was funded by the Bayerische Forschungsstiftung (AZ-1031-12).

A model for cell wall dissolution in mating yeast cells: polarized secretion and restricted diffusion of cell wall remodeling enzymes induces local dissolution.

Mating of the budding yeast, Saccharomyces cerevisiae, occurs when two haploid cells of opposite mating types signal using reciprocal pheromones and receptors, grow towards each other, and fuse to form a single diploid cell. To fuse, both cells dissolve their cell walls at the point of contact. This event must be carefully controlled because the osmotic pressure differential between the cytoplasm and extracellular environment causes cells with unprotected plasma membranes to lyse. If the cell wall-degrading enzymes diffuse through the cell wall, their concentration would rise when two cells touched each other, such as when two pheromone-stimulated cells adhere to each other via mating agglutinins. At the surfaces that touch, the enzymes must diffuse laterally through the wall before they can escape into the medium, increasing the time the enzymes spend in the cell wall, and thus raising their concentration at the point of attachment and restricting cell wall dissolution to points where cells touch each other. We tested this hypothesis by studying pheromone treated cells confined between two solid, impermeable surfaces. This confinement increases the frequency of pheromone-induced cell death, and this effect is diminished by reducing the osmotic pressure difference across the cell wall or by deleting putative cell wall glucanases and other genes necessary for efficient cell wall fusion. Our results support the model that pheromone-induced cell death is the result of a contact-driven increase in the local concentration of cell wall remodeling enzymes and suggest that this process plays an important role in regulating cell wall dissolution and fusion in mating cells.

Genetically engineered transvestites reveal novel mating genes in budding yeast.

Haploid budding yeast has two mating types, defined by the alleles of the MAT locus, MATa and MATα. Two haploid cells of opposite mating types mate by signaling to each other using reciprocal pheromones and receptors, polarizing and growing toward each other, and eventually fusing to form a single diploid cell. The pheromones and receptors are necessary and sufficient to define a mating type, but other mating-type-specific proteins make mating more efficient. We examined the role of these proteins by genetically engineering "transvestite" cells that swap the pheromone, pheromone receptor, and pheromone processing factors of one mating type for another. These cells mate with each other, but their mating is inefficient. By characterizing their mating defects and examining their transcriptomes, we found Afb1 (a-factor barrier), a novel MATα-specific protein that interferes with a-factor, the pheromone secreted by MATa cells. Strong pheromone secretion is essential for efficient mating, and the weak mating of transvestites can be improved by boosting their pheromone production. Synthetic biology can characterize the factors that control efficiency in biological processes. In yeast, selection for increased mating efficiency is likely to have continually boosted pheromone levels and the ability to discriminate between partners who make more and less pheromone. This discrimination comes at a cost: weak mating in situations where all potential partners make less pheromone.

Heterozygosity assessment of five STR loci located at 5q13 region for preimplantation genetic diagnosis of spinal muscular atrophy

Preimplantation genetic diagnosis (PGD) has been considered as an alternative to prenatal diagnosis for prevention of genetic disorders while avoiding the subsequent termination of pregnancy. However, the limited amount of template DNA available in a single diploid cell used for PGD leads to number of problems including an increased incidence of detectable contamination; amplification failure and allele drop out. Due to their highly polymorphic and amplifiable characteristics, short tandem repeat (STR) analysis has been proposed as a mean to overcome these limitations. Heterozygosity of the applied STRs is of paramount importance in their informativity, and should therefore be studied in any certain population. Here, for the first time, we report on the heterozygosity analysis of five STR markers (D5S1408, D5S1417, D5S610, D5S629 and D5S637) flanking to SMA gene region, to examine their applicability in the PGD for SMA disease. We have also investigated other statistical features of these markers and found that all of the five studied STRs were informative and four meet the Hardy-Weinberg equilibrium for the studied population. Furthermore, our results propose that similar approaches can be used for the PGD of other single gene disorders.

Cell fusion causes DNA double‐stranded breaks and transformation

Cell fusion promotes nuclear reprogramming, tissue repair and regeneration but may also induce tumors. Whether cell fusion drives tumorigenesis on its own is not known. To address this question, we fused rat intestinal epithelial cells (IEC‐6), which have a normal karyotype, exhibit contact inhibition and anchorage dependant growth, using polyethylene glycol and sorted the hybrids by FACS to establish clonal populations. Clones of single diploid IEC‐6 cells were also established as controls. After ten passages, we determined the fraction of fused clones that grew spheres in soft agar, a measure of cell transformation. More than 25% of the fusion‐derived clones were transformed, compared to 1% of non‐fused IEC‐6 clones. Fusion derived cells exhibited chromosome truncation and translocation, suggesting DNA double‐stranded breaks occurred at the outset or early passage. Consistent with this possibility, fusion‐derived cells studied at early passages had a high rate of DNA double‐stranded breaks, as estimated by γH2AX staining and comet assays. We propose that fusion‐induced DNA double‐strand breaks predispose cells to transformation by increasing the frequency of chromosomal rearrangements. These findings implicate cell fusion, as seen in inflammation, tissue injury and repair, in the genesis of tumors. This work is supported by NIH grant 5R37HL052297.

Meiotic Progression inArabidopsisIs Governed by Complex Regulatory Interactions between SMG7, TDM1, and the Meiosis I–Specific Cyclin TAM

Meiosis is a modified cell division that produces four haploid nuclei from a single diploid cell in two rounds of chromosome segregation. Here, we analyze the role of Arabidopsis thaliana SUPPRESSOR WITH MORPHOGENETIC EFFECTS ON GENITALIA7 (SMG7), THREE DIVISION MUTANT1 (TDM1), and TARDY ASYNCHRONOUS MEIOSIS (TAM) in meiotic progression. SMG7 is a conserved nonsense-mediated mRNA decay factor that is also, in Arabidopsis, essential for completion of meiosis. Examination of activating CYCLIN DEPENDENT KINASE A;1 phosophorylation at Thr-161 suggests that the meiotic arrest observed in smg7 mutants is likely caused by a failure to downregulate cyclin-dependent kinase (CDK) activity at the end of the second meiotic division. Genetic analysis indicates that SMG7 and TDM1 act in the same pathway to facilitate exit from meiosis. We further demonstrate that the cyclin TAM is specifically expressed in meiosis I and has both stimulatory and inhibitory effects on progression to meiosis II. TAM knockouts skip the second meiotic division producing unreduced gametes, but inactivation of SMG7 or TDM1 alleviates TAM's requirement for entry into meiosis II. We propose a model that meiotic progression in Arabidopsis pollen mother cells is driven by a yet to be identified cyclin-CDK activity that is modulated by regulatory interactions between TDM1, SMG7, and TAM.

Scoring of sperm chromosomal abnormalities by manual and automated approaches: qualitative and quantitative comparisons

It is now well known that levels of sperm disomy correlate to levels of infertility (as well as other factors). The risk of perpetuating aneuploidy to the offspring of infertile males undergoing intracytoplasmic sperm injection (ICSI) has become a hotly debated issue in assisted reproduction; however, there remain barriers to the practical implementation of offering sperm disomy screening in a clinical setting. The major barrier is the operator time taken to analyze a statistically meaningful (sufficient) number of cells. The introduction of automated 'spot counting' software-hardware combinations presents a potential solution to this problem. In this preliminary validation study, we analyzed 10 patients, both manually and using a commercially available spot counter. Results show a statistically significant correlation between both approaches for scoring of sperm disomy, but no correlation is found when scoring for diploid sperm. The most likely explanation for the latter is an apparent overscoring of two closely associated sperm heads as a single diploid cell. These results, and similar further studies that will ensue, help to inform cost-benefit analyses that individual clinics need to carry out in order to decide whether to adopt sperm aneuploidy screening as a routine tool for the assessment of sperm from men requiring ICSI treatment.

A single nucleotide polymorphism melt curve assay employing an intercalating dye probe fluorescence resonance energy transfer for forensic analysis

The characterization and use of DNA sequence polymorphisms are an important aspect of forensic analysis. A number of approaches are being explored for single nucleotide polymorphism (SNP) genotyping, but current detection methods are subject to limitations that adversely impact their utility for forensic analysis. We have developed a novel method for genotyping both single and multiple SNPs that uses an intercalating dye and a probe labeled with a single fluorophore to affect a fluorescence energy transfer. Melting curve analysis is then used to distinguish true alleles from mismatched alleles. We term the new method dye probe fluorescence resonance energy transfer (dpFRET). In the current work, development proceeded at first with synthetic DNA template testing to establish proof of concept for the chemistry involved, followed by the design of polymerase chain reaction (PCR)-based genomic DNA assays to demonstrate potential forensic applications. The loci chosen for testing included both nuclear (MHC DRB) and mitochondrial DNA (cytochrome b) genes. A preliminary assessment of the sensitivity limits of the technology indicated that dpFRET was capable of accurately genotyping DNA from one single diploid cell equivalent. This technology could also potentially impact a wide range of nonforensic disciplines to aid in discovery, screening, and association of DNA sequence polymorphisms.

Ploidy chimeras induced in haploid sporophytes of Osmunda claytoniana and Osmunda japonica

Haploid sporophytes of Osmunda claytoniana (2n = x = 22) were apogamously produced from calli when cultivated on a hormone-free medium. Flow cytometric analysis showed that ploidy chimeras were spontaneously produced in a haploid sporophyte of O. claytoniana and those of O. japonica that were obtained in the previous study. In the haploid sporophyte of O. claytoniana, a diploid pinnule and a partially diploid terminal segment were produced in a haploid pinna. In O. japonica, a haploid sporophyte yielded a diploid pinna in a haploid frond, and another haploid sporophyte yielded a diploid pinnule in a haploid pinna. Diploid chimeras were large in size and could be readily distinguished from other haploid parts of the fronds. It is likely that the chimeras were produced clonally from a single diploid cell that established chromosome doubling.