Ribosomal RNA synthesis and processing in a particulate site in the HeLa cell nucleus

A particulate fraction has been isolated from detergent-prepared HeLa cell nuclei. The fraction consists largely of organelles that resemble the nucleoli of intact cells. The 45S RNA that is precursor to 28S and 18S ribosomal RNA is associated with the fraction. The 32S RNA that is labeled after the 45S RNA and is the apparent precursor to 28S RNA is also associated with the fraction. The nucleoplasm contains 28S RNA that behaves as an intermediate between the 32S nucleolar RNA and the 28S cytoplasmic RNA.     

Enzymatic modification of transfer RNA

The molecular events leading to the synthesis of mature tRNA are only now becoming amenable to experimental study. In bacterial and mammalian cells tRNA genes are transcribed into precursor tRNA. These molecules, when isolated, contain additional nucleotides at both ends (20) of the mature tRNA and lack most modified nucleosides. Presumably, specific nucleases ("trimming" enzymes) cut the precursor to proper tRNA size. The C-C-A nucleotide sequence of the amino acid acceptor end common to all tRNA's does not seem to be coded by tRNA genes (30), and may be added to the trimmed molecules by the tRNA-CMP-AMP-pyrophosphorylase (71). Modifications at the polynucleotide level of the heterocyclic bases or the sugar residues give rise to the modified nucleosides in tRNA. Although newly available substrates have allowed the detection of more of the enzymes involved in these reactions, there is still no knowledge about the sequence of modification or trimming events leading to the synthesis of active tRNA. Progress in these studies may not be easy because enzyme preparations free of nucleases or other tRNA modifying enzymes are required. The role of the modified nucleosides in the biological functions of tRNA is still unknown. Possibly pseudouridine is required for ribosome mediated protein synthesis; some other modified nucleosides in tRNA are not required for this reaction, but may enhance its rate. What might be the role of the large variety of modified nucleosides in tRNA? One is tempted to speculate that such nucleosides are important in other cellular processes in which tRNA is thought to participate such as virus infection, cell differentiation, and hormone action (2, 3). Mutants in a number of tRNA-modifying enzymes are needed in order to extend our knowledge of their purpose and of tRNA involvement in other biological processes. But unless tRNA-modifying enzymes specific for a particular tRNA species exist, no simple selection procedure can be devised. Possibly some of the regulatory mutants of amino acid biosynthesis may prove to affect tRNA-modifying enzymes (72). Transfer RNA's are macromolecules well suited for the study of nucleic acid-protein interactions. The tRNA molecules are structurally very similar, and they interact with a large number of enzymes or protein factors (2, 3). Each aminoacyl-tRNA synthetase, for instance, very precisely recognizes a set of cognate isoacceptor tRNA's (2, 73). The availability of the tRNA- modifying enzymes adds another dimension to the problem of the nature of specific recognition of tRNA by proteins. There are some tRNA-modifying enzymes, such as the uracil-tRNA methylase, which may recognize all tRNA species, while others, such as the isopentenyl-tRNA transferase, probably recognize only a selected set of tRNA molecules, even with different amino acid accepting capacities. With well-characterized RNA precursor and tRNA molecules we can hope to delineate those features of primary, secondary, and tertiary structure involved in the specific interactions of tRNA with these enzymes.     

Extrachromosomal microDNAs and chromosomal microdeletions in normal tissues

We have identified tens of thousands of short extrachromosomal circular DNAs (microDNA) in mouse tissues as well as mouse and human cell lines. These microDNAs are 200 to 400 base pairs long, are derived from unique nonrepetitive sequence, and are enriched in the 5'-untranslated regions of genes, exons, and CpG islands. Chromosomal loci that are enriched sources of microDNA in the adult brain are somatically mosaic for microdeletions that appear to arise from the excision of microDNAs. Germline microdeletions identified by the "Thousand Genomes" project may also arise from the excision of microDNAs in the germline lineage. We have thus identified a previously unknown DNA entity in mammalian cells and provide evidence that their generation leaves behind deletions in different genomic loci.     

The maternal nucleolus is essential for early embryonic development in mammals

With fertilization, the paternal and maternal contributions to the zygote are not equal. The oocyte and spermatozoon are equipped with complementary arsenals of cellular structures and molecules necessary for the creation of a developmentally competent embryo. We show that the nucleolus is exclusively of maternal origin. The maternal nucleolus is not necessary for oocyte maturation; however, it is necessary for the formation of pronuclear nucleoli after fertilization or parthenogenetic activation and is essential for further embryonic development. In addition, the nucleolus in the embryo produced by somatic cell nuclear transfer originates from the oocyte, demonstrating that the maternal nucleolus supports successful embryonic development.     

棉花和小叶杨小孢子母细胞中超数核仁的形成及动态变化

用光镜和透射电镜观察研究了棉花(Gossypium hirsutum)和小叶杨(Populussimonii)小孢子母细胞中超数核仁的发生和动态变化过程。超数核仁出现于减数分裂前(棉花)和减数分裂前期Ⅰ早期(小叶杨),产生部位是核仁组织者区域。超数核仁与主核仁有类似的染色特性,但体积小,数量丰富。在小孢子母细胞减数分裂过程中,超数核仁通过下述主要方式进入细胞质:一是在分裂中期和后期核膜暂时解体时进入细胞质中;另一是在核膜完整时通过状态的变化经核膜孔进入细胞质或以整体形式通过核膜外突并缢出进入细胞质中。小孢子母细胞中超数核仁的发生可能是大量迅速补给细胞质核糖体数量的一种机制,与孢子体转向配子体的发育有关。     

Activin A特异性对人胚胎干细胞向限定性内胚层诱导分化的促进作用

【目的】研究Activin A对人胚胎干细胞（hESCs）向限定性内胚层（DE）诱导分化的促进作用及其信号通路分子,为hESCs向DE诱导分化体系的优化提供参考。【方法】在人饲养层体系培养的hESCs中,收集100 ng/mL Activin A分别诱导0,6,12,24,48,72,96,120 h的细胞,用实时荧光定量RT-PCR检测原条标记Gsc和Mixl1、中内胚层共同前体标记Brachyury、内胚层标记Foxa2和Sox17、中胚层标记Flk1、外胚层基因Pax6、多能性相关基因Oct4与Nanog表达水平的变化,用细胞免疫荧光检测Brachyury和Sox17蛋白表达水平的变化。【结果】在人饲养层（HEF）培养体系上,高浓度Activin A能更快地促进中内胚层基因的表达并提高其表达水平;Brachyury和Sox17蛋白的细胞免疫荧光检测表明,Activin A诱导12和48 h就可检测二者的表达明显增加,且二者的表达水平分别在诱导48和96 h时达到高峰;hESCs高效分化为限定性内胚层细胞,DE细胞分化率为（81.7±5.4）%,并且体外的内胚层分化过程遵循从原条开始、经过中内胚层共同前体阶段、再到内胚层的发育过程,与体内发育规律相似。【结论】Activin A能特异性地诱导人胚胎干细胞向限定性内胚层分化,转录调控Brachyury和Sox17蛋白的表达。     

Quiescent T lymphocytes as an inducible virus reservoir in HIV-1 infection

To better understand the basis for human immunodeficiency virus type 1 (HIV-1) persistence and latency, the form in which viral DNA exists in the peripheral T lymphocyte reservoir of infected individuals was investigated. In asymptomatic individuals, HIV-1 was harbored predominantly as full-length, unintegrated complementary DNA. These extrachromosomal DNA forms retained the ability to integrate upon T cell activation in vitro. In patients with acquired immunodeficiency syndrome (AIDS), there was an increase in integrated relative to extrachromosomal DNA forms. By analysis of DNA from patient lymphocyte subpopulations depleted of human lymphocyte antigen-Dr receptor-positive cells, quiescent T cells were identified as the source of extrachromosomal HIV-1 DNA. Thus quiescent T lymphocytes may be a major and inducible HIV-1 reservoir in infected individuals.     

奶牛乳腺上皮细胞的原代培养

1材料与方法 1．1材料 1．1．1乳腺组织。从屠宰场选取健康的泌乳期奶牛,无菌手术切取乳腺实质组织,置于DMEM／F12完全培养液（含10％胎牛血清、100IU／ml青霉素和链霉素）中,尽快运回实验室。     

Stable amplified DNA in drug-resistant Leishmania exists as extrachromosomal circles

The relative stability of amplified DNA in drug-resistant Leishmania major was previously reported to be dependent on location, that is, unstable amplified DNA was extrachromosomal and stable amplified DNA was chromosomal. Leishmanial chromosomes have now been directly examined by means of orthogonal-field-alternation gel electrophoresis (OFAGE). The amplified DNA's in three resistant cell lines displayed unusual migration and were clearly extrachromosomal, regardless of whether the amplified DNA's were stable or unstable. Thus, contrary to conclusions from earlier studies of drug resistance in cultured animal cells, stable amplified DNA in Leishmania can be extrachromosomal. In addition, these amplified DNA's were shown to be circular on the basis of their resistance to exonuclease III digestion and their behavior on OFAGE. Their mobility was also greatly changed after treatment with topoisomerase II, suggesting that the amplified DNA's were either supercoiled or concatenated circles.     

Transfectomas provide novel chimeric antibodies

Methods have been developed to transfect immunoglobulin genes into lymphoid cells. The transfected genes are faithfully expressed, and assembly can occur both between the transfected and endogenous chains and between two transfected chains. Gene transfection can be used to reconstitute immunoglobulin molecules and to produce novel immunoglobulin molecules. These novel molecules can represent unique combinations of heavy and light chains; alternatively, by means of recombinant DNA technology, genes can be assembled in vitro, transfected, and expressed. The end products of such manipulations include chimeric molecules with variable regions joined to different isotypic constant regions; this is possible both within and between species. It is also possible to synthesize altered immunoglobulin molecules, as well as molecules having immunoglobulin sequences fused with nonimmunoglobulin sequences (for example, enzyme sequences).     

Drosophila CENH3 is sufficient for centromere formation

CENH3 is a centromere-specific histone H3 variant essential for kinetochore assembly. Despite its central role in centromere function, there has been no conclusive evidence supporting CENH3 as sufficient to determine centromere identity. To address this question, we artificially targeted Drosophila CENH3 (CENP-A/CID) as a CID-GFP-LacI fusion protein to stably integrated lac operator (lacO) arrays. This ectopic CID focus assembles a functional kinetochore and directs incorporation of CID molecules without the LacI-anchor, providing evidence for the self-propagation of the epigenetic mark. CID-GFP-LacI-bound extrachromosomal lacO plasmids can assemble kinetochore proteins and bind microtubules, resulting in their stable transmission for several cell generations even after eliminating CID-GFP-LacI. We conclude that CID is both necessary and sufficient to serve as an epigenetic centromere mark and nucleate heritable centromere function.     

Cassette of eight exons shared by genes for LDL receptor and EGF precursor

The amino acid sequences of the human low-density lipoprotein (LDL) receptor and the human precursor for epidermal growth factor (EGF) show 33 percent identity over a stretch of 400 residues. This region of homologous is encoded by eight contiguous exons in each respective gene. Of the nine introns that separate these exons, five are located in identical positions in the two protein sequences. This finding suggests that the homologous region may have resulted from a duplication of an ancestral gene and that the two genes evolved further by recruitment of exons from other genes, which provided the specific functional domains of the LDL receptor and the EGF precursor.     

DNA replication-independent silencing in S. cerevisiae

In Saccharomyces cerevisiae, the silent mating loci are repressed by their assembly into heterochromatin. The formation of this heterochromatin requires a cell cycle event that occurs between early S phase and G(2)/M phase, which has been widely assumed to be DNA replication. To determine whether DNA replication through a silent mating-type locus, HMRa, is required for silencing to be established, we monitored heterochromatin formation at HMRa on a chromosome and on a nonreplicating extrachromosomal cassette as cells passed through S phase. Cells that passed through S phase established silencing at both the chromosomal HMRa locus and the extrachromosomal HMRa locus with equal efficiency. Thus, in contrast to the prevailing view, the establishment of silencing occurred in the absence of passage of the DNA replication fork through or near the HMR locus, but retained a cell cycle dependence.     

Curvilinear, three-dimensional motion of chromatin domains and nucleoli in neuronal interphase nuclei

The term "nuclear rotation" refers to a motion of nucleoli within interphase nuclei of several cell types. No mechanism or function has been ascribed to this phenomenon, and it was unknown whether nuclear structures in addition to nucleoli participate in this motion. Moreover, it was unclear whether nuclear rotation occurs independent of concurrent motion of juxtanuclear cytoplasm. The work reported here presents quantitative evidence, for three-dimensional intranuclear, tandem motion of fluorescently labeled chromatin domains associated with nucleoli and those remote from nucleoli. The results show that such motion is curvilinear, that it is not restricted to nucleoli, and, moreover, that it occurs independently of motion of juxtanuclear, cytoplasmic structures. These results suggest that this motion represents karyoplasmic streaming and its function is to transpose to nuclear pores those chromatin domains actively transcribed.     

Permuted tRNA genes expressed via a circular RNA intermediate in Cyanidioschyzon merolae

A computational analysis of the nuclear genome of a red alga, Cyanidioschyzon merolae, identified 11 transfer RNA (tRNA) genes in which the 3' half of the tRNA lies upstream of the 5' half in the genome. We verified that these genes are expressed and produce mature tRNAs that are aminoacylated. Analysis of tRNA-processing intermediates for these genes indicates an unusual processing pathway in which the termini of the tRNA precursor are ligated, resulting in formation of a characteristic circular RNA intermediate that is then processed at the acceptor stem to generate the correct termini.     

Synthesis, transport, and release of posterior pituitary hormones

Vasopressin and oxytocin are made and released by neurons of the hypothalamo-neurohypophysial system. Pulse labeling these neurons with radioactive amino acid indicates that the two hormones and their respective neurophysin carrier proteins are synthesized as parts of separate precursor proteins. The precursors seem to be processed into smaller, biologically active molecules while they are being transported along the axon.     

Chromosomal and nucleolar RNA synthesis in root tips during mitosis

Comparative rates of RNA synthesis in chromatin and nucleolar fractions during mitosis in root-tip cells of Allium and Nigella were studied by pulse-labeling of cells with tritiated cytidine. Although the rate of RNA synthesis decreases in the condensing chromosomes during prophase, it remains normal in the nucleolar fraction as long as nucleoli are maintained. RNA synthesis stops in mitotic cells lacking distinct nucleoli. In the late telophase or very early interphase cells, RNA synthesis resumes at a faster rate in the pronucleolar bodies than in the chromatin.     

Amyloid beta protein precursor is possibly a heparan sulfate proteoglycan core protein

The amyloid beta protein peptide is a major constituent of amyloid plaque cores in Alzheimer's disease and is apparently derived from a higher molecular weight precursor. It is now shown that the core protein of a heparan sulfate proteoglycan secreted from a nerve cell line (PC12) has an amino acid sequence and a size very similar to those of the amyloid beta protein precursor and that these molecules are antigenically related. This amyloid beta protein precursor-related protein is not found in the conditioned medium of a variant cell line (F3 PC12) that does not secrete heparan sulfate proteoglycan. The synaptic localization and metabolism of this class of proteoglycans are consistent with its potential involvement in central nervous system dysfunction.     

Selective cleavage of D-Ala-D-Lac by small molecules: re-sensitizing resistant bacteria to vancomycin

Pathogenic enterococci are becoming resistant to currently available antibiotics, including vancomycin, the drug of last resort for Gram-positive infections. Enterococci pose a significant public health threat, not least because of the risk of transferring vancomycin resistance to the ubiquitous Staphylococcus aureus. Vancomycin resistance is manifested by cell wall peptidoglycan precursors with altered termini that cannot bind the antibiotic. Small molecules with well-oriented nucleophile-electrophile assembly and complementary chirality to the peptidoglycan termini were identified as catalytic and selective cleavers of the peptidoglycan precursor depsipeptide. These molecules were tested in combination with vancomycin and were found to re-sensitize vancomycin-resistant bacteria to the antibiotic.     

The chemistry of self-splicing RNA and RNA enzymes

Proteins are not the only catalysts of cellular reactions; there is a growing list of RNA molecules that catalyze RNA cleavage and joining reactions. The chemical mechanisms of RNA-catalyzed reactions are discussed with emphasis on the self-splicing ribosomal RNA precursor of Tetrahymena and the enzymatic activities of its intervening sequence RNA. Wherever appropriate, catalysis by RNA is compared to catalysis by protein enzymes.