朱顶红精细胞超微结构观察

精细胞是双受精作用的直接参与者,是生殖生物学中的重点研究对象之一。以往的研究表明,应用连续超薄切片和计算机辅助三维重组技术,结合免疫荧光定位,发现两个精细胞在体积和细胞器含量上存在着差异,即精子的二型性,而且与营养细胞核三者构成紧密功能单位即雄性生殖单位（ＭＧＵ）,微管对精细胞的性状的确定、运动和维持ＭＧＵ的动态结构稳定具有重要的作用。本文应用透射电镜,详细观察了朱顶红花粉管中精细胞的超微结构,并着重微管结构及其分布的观察。朱顶红成熟花粉为两细胞型。成熟花粉于２６℃、黑暗条件下,在液体培养基（含１０％蔗糖和１００ｐｐｍ硼酸）中培养１３－１８小时,然后收集花粉管,固定,供电镜观察并照相。朱顶红成熟花粉培养１３小时后,生殖细胞在花粉管中完成核分裂和胞质分裂等两个过程。形成两个精细胞。初形成的两个精细胞前后排列,营养核前导并靠近花粉管顶端。领头的精细胞的细胞质以很大的表面与营养核相互贴合（图版Ⅰ－１,２）,有时营养核与两个精细胞彼此穿插、缠绕（图版Ⅰ－３）。两精细胞之间的共同壁上具有很多胞质通道和含均质电子密度中等的基质（图版Ⅱ－４）。精细胞质在核与共同壁之间的区域染色较深,经高倍放大,观察到此处含丰富的微管,基     

Nuclear architecture and DNA location in two VAM fungi

Summary The ultrastructural organization of nuclei in Glomus versiforme and G. mosseae hyphae was examined by sampling at different times during the fungus life cycle. In young and active hyphae, the nucleus is chromocentric with dispersed chromatin and a well-developed nucleolus. On aging, and particularly during arbuscule collapse, the chromatin condenses to give a pyk-notic nucleus with blebs in the envelope and conspicuous spaces between this and the nucleoplasm. DNA location by immunogold or enzyme-gold techniques resulted in uniform labelling over the chromatin. Nuclear degeneration is mirrored by decreased gold labelling and may thus be regarded as an important parameter for evaluation of fungus viability and the ephemeral life of the arbuscule.     

The molecular organizational characteristics of the cell nucleus components at different phases of the mitotic cycle and in the resting state]

Data about the changes of the cell nucleus structure at different levels of its organization are summarized in the review. The data about the change of the DNA break number during the cycle and in resting state are presented and the role of the changes of the repair efficiency in this process is discussed. The changes of the chromatin protein spectrum, the chromatin structure at nucleosomal and supranucleosomal levels, the DNA superhelicity, topoisomerase activity, nuclear matrix composition and structure are discussed as well. The nucleus structure during the S-phase and mitosis and the cycle-related changes of the chromatin structure in lower eukaryotes are reviewed separately.     

Compartmentalization of regulatory proteins in the cell nucleus

The cell nucleus is increasingly recognized as a spatially organized structure. In this review, the nature and controversies associated with nuclear compartmentalization are discussed. The relationship between nuclear structure and organization of proteins involved in the regulation of RNA polymerase II-transcribed genes is then discussed. Finally, very recent data on the mobility of these proteins within the cell nucleus is considered and their implications for regulation through compartmentalization of proteins and genomic DNA are discussed.     

Mechanisms of single-stranded DNA-binding protein functioning in cellular DNA metabolism

This review deals with analysis of mechanisms involved in coordination of DNA replication and repair by SSB proteins; characteristics of eukaryotic, prokaryotic, and archaeal SSB proteins are considered, which made it possible to distinguish general mechanisms specific for functioning of proteins from organisms of different life domains. Mechanisms of SSB protein interactions with DNA during metabolism of the latter are studied; structural organization of the SSB protein complexes with DNA, as well as structural and functional peculiarities of different SSB proteins are analyzed.     

Review: The Dynamics of the Nuclear Lamins during the Cell Cycle— Relationship between Structure and Function

The nuclear lamins are members of the intermediate filament (IF) family of proteins. The lamins have an essential role in maintaining nuclear integrity, as do the other IF family members in the cytoplasm. Also like cytoplasmic IFs, the organization of lamins is dynamic. The lamins are found not only at the nuclear periphery but also in the interior of the nucleus, as distinct nucleoplasmic foci and possibly as a network throughout the nucleus. Nuclear processes such as DNA replication may be organized around these structures. In this review, we discuss changes in the structure and organization of the nuclear lamins during the cell cycle and during cell differentiation. These changes are correlated with changes in nuclear structure and function. For example, the interactions of lamins with chromatin and nuclear envelope components occur very early during nuclear assembly following mitosis. During S-phase, the lamins colocalize with markers of DNA replication, and proper lamin organization must be maintained for replication to proceed. When cells differentiate, the expression pattern of lamin isotypes changes. In addition, changes in lamin organization and expression patterns accompany the nuclear alterations observed in transformed cells. These lamin structures may modulate nuclear function in each of these processes.     

Machine Learning Methods for Exploring Sequence Determinants of 3D Genome Organization

In higher eukaryotic cells, chromosomes are folded inside the nucleus. Recent advances in whole-genome mapping technologies have revealed the multiscale features of 3D genome organization that are intertwined with fundamental genome functions. However, DNA sequence determinants that modulate the formation of 3D genome organization remain poorly characterized. In the past few years, predicting 3D genome organization based on DNA sequence features has become an active area of research. Here, we review the recent progress in computational approaches to unraveling important sequence elements for 3D genome organization. In particular, we discuss the rapid development of machine learning-based methods that facilitate the connections between DNA sequence features and 3D genome architectures at different scales. While much progress has been made in developing predictive models for revealing important sequence features for 3D genome organization, new research is urgently needed to incorporate multi-omic data and enhance model interpretability, further advancing our understanding of gene regulation mechanisms through the lens of 3D genome organization.     

Molecular and chromosomal organization of two repetitive DNA sequences with intercalary locations in sugar beet and other Beta species

 In a search for repetitive DNA sequences in the sugar beet genome, two sequences with repeat unit lengths of 143 and 434 bp were isolated and characterized. The pSV family showed an unusual conservation of restriction sites reflecting homogenization of the analyzed repeats. Members of the family are organized as tandem repeats as revealed by PCR and sequencing of dimeric units. The pSV satellite occurs in large intercalary arrays which are present on all chromosome arms of sugar beet. The pSV sequence family is present in different abundance in the sections Beta, Corollinae and Nanae but is not detectable by Southern hybridization in the section Procumbentes. The pDRV family is characterized by an interspersed genomic organization. The sequence is detectable in all sections of the genus and is amplified in species of the section Beta but was also detected, although at lower abundance, in the remaining three sections. Fluorescent in situ hybridization has shown that the pDRV sequence family is dispersed over all chromosomes of the sugar beet complement with some regions of clustering and centromeric depletion. Received: 18 March 1998 / Accepted: 31 March 1998     

Linker histones: conformational changes and the role in the structural organization of chromatin

Histones, linker histones of the H1 family, their postsyntetic modifications, DNA-histone H1 interaction are reviewed. A question of protein change in spermatogenesis at the formation of inactive nucleus with high degree of DNA density is considered. Special attention was paid to sperm-specific histones of the H1 family of sperm cells. Their role in organization of high-order chromatin structure of sperm cells is discussed. Also, results of different studies on the structural organization of chromatin (nucleosomes, 30-nm fibers, chromatin loops and metaphase chromosomes) are discussed.     

Der Zellkern – eine Stadt in der Zelle

Nuclear architecture Stored genetic information is useless, if it cannot be retrieved at the right time and the right place. Packaging of DNA within the chromatin and dynamic changes of its spatiotemporal arrangements of the cell nucleus have a fundamental impact on gene expression and other nuclear functions. In this review the authors describe the development of experimental research on nuclear architecture and of corresponding changes of concepts about the functional organization of the cell nucleus.     

核基质附着区广泛参与基因组三维结构折叠的生物信息学分析

在细胞分裂间期,每条染色质都占据着特定的染色质领域（chromosome territory,CT）。每个CT领域内进一步分成不同的拓扑学相关区域（topological associated domain,TAD）,每个TAD又由若干子TAD（sub-TAD）构成。不同的TAD相互聚集,形成基因活跃表达和不表达的A、B两种组份或区室（compartment）。然而,目前对于染色质折叠方式及维持机制的研究尚无定论。核基质附着区（matrix attachment regions,MARs）是在不同物种基因组中广泛存在的一类富含AT序列的与核基质结合的DNA元件,能够通过与CTCF、SATB1等调控蛋白质相互作用,对远距离的基因表达进行调控。本研究以染色质三维结构为背景,通过整合染色质三维结构及组蛋白修饰等组学数据,对MARs元件与染色质三维结构的关系进行研究,对MARs元件参与形成的相互作用网络的结构及功能进行探索。结果发现,MARs元件与染色质三维结构高度相关,而且在高强度相互作用中占据较大的比例,提示MARs元件在染色质折叠方面发挥作用。此外,通过拓扑结构聚类分析还首次揭示,MARs元件分为不同类型,包括维持染色质领域及空间构象等的结构单元部分,以及调控基因表达等的功能单元部分。这表明,MARs元件在基因组三维高级结构的建立、维持以及功能等方面发挥重要作用。     

Preferential nuclear location of a transgene does not depend on its transcriptional activity during early mouse development

Changes in chromatin structure play an important role in regulation of the HSP70.1 gene during mouse preimplantation development. Using in situ PCR we have now examined whether the spatial organization of an HSP70.1 luciferase transgene within the nucleus is also a factor in regulating its expression. The transgene showed a preferential localization towards the nuclear periphery throughout preimplantation development. This preferential location was independent of the level of constitutive activity of the transgene and did not change when transgene expression was induced through core histone hyperacetylation at the eight-cell stage or by heat shock in blastocysts. In contrast, at the two-cell stage, when embryos are unable to continue development after heat shock, thermal stress provoked a significant disruption of the nuclear location of the transgene. These results do not agree with a recent model of embryonic genome activation in mice which hypothesizes that directed, active movement of DNA within the nucleus is a determinant factor in establishing early patterns of gene expression. Instead, they are consistent with models proposing that chromatin segments are restricted to nuclear subregions, but that they remain free to undergo substantial Brownian motion. Received: 21 May 1998; in revised form: 21 July 1998 / Accepted: 21 July 1998     

The Long and Viscous Road: Uncovering Nuclear Diffusion Barriers in Closed Mitosis

Diffusion barriers are effective means for constraining protein lateral exchange in cellular membranes. In Saccharomyces cerevisiae, they have been shown to sustain parental identity through asymmetric segregation of ageing factors during closed mitosis. Even though barriers have been extensively studied in the plasma membrane, their identity and organization within the nucleus remains poorly understood. Based on different lines of experimental evidence, we present a model of the composition and structural organization of a nuclear diffusion barrier during anaphase. By means of spatial stochastic simulations, we propose how specialised lipid domains, protein rings, and morphological changes of the nucleus may coordinate to restrict protein exchange between mother and daughter nuclear lobes. We explore distinct, plausible configurations of these diffusion barriers and offer testable predictions regarding their protein exclusion properties and the diffusion regimes they generate. Our model predicts that, while a specialised lipid domain and an immobile protein ring at the bud neck can compartmentalize the nucleus during early anaphase; a specialised lipid domain spanning the elongated bridge between lobes would be entirely sufficient during late anaphase. Our work shows how complex nuclear diffusion barriers in closed mitosis may arise from simple nanoscale biophysical interactions.     

Domain organization and biochemical features of Sulfolobus solfataricus DNA polymerase

DNA polymerase from Sulfolobus solfataricus, strain MT4 (Sso DNA pol), was one of the first archaeal DNA polymerases to be isolated and characterized. Its encoding gene was cloned and sequenced, indicating that Sso DNA pol belongs to family B of DNA polymerases. By limited proteolysis experiments carried out on the recombinant homogeneous protein, we were able to demonstrate that the enzyme has a modular organization of its associated catalytic functions (DNA polymerase and 3′-5′ exonuclease). Indeed, the synthetic function was ascribed to the enzyme C-terminal portion, whereas the N-terminal half was found to be responsible for the exonucleolytic activity. In addition, partial proteolysis studies were utilized to map conformational changes on DNA binding by comparing the cleavage map in the absence or presence of nucleic acid ligands. This analysis allowed us to identify two segments of the Sso DNA pol amino acid chain affected by structural modifications following nucleic acid binding: region 1 and region 2, in the middle and at the C-terminal end of the protein chain, respectively. Site-directed mutagenesis studies will be performed to better investigate the role of these two protein segments in DNA substrate interaction. Received: January 22, 1998 / Accepted: February 16, 1998     

Formation of DNA replication structures in herpes virus-infected cells requires a viral DNA binding protein

Eukaryotic DNA synthesis is thought to occur in multienzyme complexes present at numerous discrete sites throughout the nucleus. We demonstrate here that cellular DNA replication sites identified by bromodeoxyuridine labeling are relocated in cells infected with herpes simplex virus such that they correspond to viral prereplicative structures containing the HSV DNA replication protein, ICP8. Thus components of the cellular DNA replication apparatus are present at viral prereplicative sites. Mutant virus strains expressing defective ICP8 do not alter the pattern of host cell DNA replication sites, indicating that functional ICP8 is required for the redistribution of cellular DNA replication complexes. This demonstrates that a specific protein molecule can play a role in the organization of DNA replication proteins at discrete sites within the cell nucleus.     

A phosphoinositide-binding sequence is shared by PH domain target molecules—a model for the binding of PH domains to proteins

Pleckstrin homology (PH) domains have been proven to bind phosphoinositides (PI) and inositolphosphates (IP). On the other hand, a binding of PH domains to proteins is still a matter of debate. The goal of this work was to identify potential PH domain protein target sites and to build a model for PH domain–protein binding. A candidate sequence, called HIKE, was identified by sequence homology analysis of the proteins that are considered the strongest PH binding candidates, i.e., G_β, PKC, and Akt. HIKE contains a PI binding sequence and fulfills several criteria for a potential PH-binding site, i.e., it is present in other PH-binding candidates, lies in regulatory regions independently predicted to bind PH domains, and is conserved in 3-D structure among different molecules. These findings and the similarities with the mode of binding of PTB and PDZ domains suggest a β strand–β strand coordination model for PH–protein binding. The HIKE model predicts that membrane anchoring of PH domains and their targets could be a critical step in their interaction, which would consistently explain why PH–protein binding has only been detected in the presence of PI. Proteins 31:1–9, 1998. © 1998 Wiley-Liss, Inc.     

Glial organization and chondroitin sulfate proteoglycan expression in the developing thalamus

This study examines the early organization of glial cells, together with the expression of chondroitin sulfate proteoglycans in the developing thalamus of ferrets. Glia were identified with antibodies against vimentin and glial fibrillary acidic protein and the chondroitin sulfate proteoglycans were identified by using an antibody against chondroitin sulfate side chains. Our results reveal three striking features of early thalamic development. First, there is a distinct population of glial fibrillary acidic protein-immunoreactive astrocytes (first seen at E30) that resides in the perireticular thalamic nucleus of the primordial internal capsule. These glial fibrillary acidic protein-immunoreactive astrocytes of the perireticular nucleus are transient and form a conspicuous feature of the early developing forebrain. They are first apparent well before any glial fibrillary acidic protein-immunoreactive astrocytes are seen in other regions of the thalamus (at about P8). Further, unlike in other thalamic regions, these peculiar perireticular astrocytes do not express vimentin before they express glial fibrillary acidic protein. Second, in the reticular thalamic nucleus, the radial glial cells express glial fibrillary acidic protein; they are the only ones to do so in the thalamus during development. The glial fibrillary acidic protein-immunoreactive radial glial cells of the reticular nucleus form a rather distinct band across the developing thalamus at these early stages (E30–P1). Finally, and preceding the expression of glial fibrillary acidic protein, the radial glial cells of the reticular nucleus, unlike those in other thalamic regions, are associated closely with the expression of chondroitin sulfate proteoglycans (E20–E30). Later (after E30), the expression of the chondroitin sulfate proteoglycans in the reticular nucleus declines sharply. The significance of this finding is related to the early organization of the cortico-fugal and cortico-petal pathways.     

Postnatal cell proliferation and death in a lateralized,gender‐related,asymmetric nucleus

Sexual differentiation and lateralization of neurone number in a discrete forebrain nucleus (SDApc) related to masculine vocal emission, occur contemporaneously in postnatal (P0–P15) gerbils. Stereological estimates of cell proliferation and death during SDApc organization were made by BrdU labelling and pyknosis, respectively. Results confirmed that rates of apoptosis were greater in females and lateralized in males. Immunoreactive BrdU cells, located in the SDApc at P0–P6, with low levels at P15, were not numerically different between the sexes. Only at one age, P0, in males, was a left‐right difference seen in BrdU‐immunoreactive cell numbers. Microglia, identified by isolectin immunostaining, were numerically similar to BrdU cells. We suggest that apoptosis, rather than neurogenesis, differentiates and lateralizes SDApc organization, and proliferating cells are microglia, phagocytosing debris. © 2001 John Wiley & Sons, Inc. J Neurobiol 47: 150–158, 2001