End Of Primer Research Articles

Cis-Acting Element-specific Transcriptional Activity of Differentially Phosphorylated Nuclear Factor-κB

Phosphorylation of nuclear factor-kappa B (NF-kappa B) subunits emerges as a mechanism by which transcriptional activity of nuclear NF-kappa B complexes is regulated in an inhibitor kappa B-independent fashion. As the main transactivator, the p65 subunit of NF-kappa B has an outstanding position in the hierarchy of NF-kappa B proteins. p65 is a multiply phosphorylated protein with phosphorylation sites in the C-terminal transactivation domain and the N-terminal Rel homology domain (RHD). In this study, we describe two previously non-reported phospho-acceptor sites within the p65 RHD. We show that differential phosphorylation of serine residues within the RHD modulates transcriptional activity in a cis-acting element and promoter-specific context, thus leading to a phosphorylation state-dependent gene expression profile. RelA(-/-) mouse embryonic fibroblasts reconstituted with wild-type p65 or p65 phosphorylation-deficient mutants showed a distinctive expression profile of synthetic kappa B-dependent reporters as well as endogenous genes. Hypophosphorylated p65 did not display cis-acting element-specific changes in DNA binding or dimerization behavior. This study shows for the first time that site-specific phosphorylation can target a transcription factor to a particular subset of genes.

Disruption of the NDE1 Gene Occurs in 90% of the Cases of inv(16) AML and Results in Chromosomal Instability and Myeloproliferative Disease in Mice.

The inv(16) chromosomal aberration is found in 10% of the cases with AML. The resultant CBFB-MYH11 fusion gene that plays a key-role in the leukemia development, but for full transformation, additional genetic hits are necessary. We report that the MYH11 gene overlaps with the NDE1 gene. At their 3 prime ends the genes overlap at the genomic, transcript as well as coding level and as a consequence, the inv(16) disrupts NDE1 in addition to CBFB and MYH11 in 90% of CBFB-MYH11 positive cases. To determine the relevance of Nde1 function in hematopoiesis leukemia development, hematopoiesis was studied in Nde1-/− and Nde1+/− mutant mice. A significant percentage of the Nde1 null mutant mice developed a myeloproliferative (MPD) phenotype at 6–12 months of age. This phenotype was observed in 6 out of 15 (40%) Nde1−/− mice, 2 out of 15 (13%) Nde1+/− mice and none of 4 wild type mice. All mice with the MPD phenotype exhibited a profound splenomegaly with spleen sizes ranging from 5 to 20-fold larger than normal wild type controls. Histopathological analysis demonstrated hypercellular bone marrow, effacement of normal splenic architecture with expansion of a myeloid cells, and infiltration of proliferating myeloid cells in various organs . A 10-fold increase in Gr1+/Mac1+ double positive mature myeloid cells in the spleen was confirmed by flow-cytometric analysis. Peripheral blood analyses showed that the white blood cell counts were slightly elevated in Nde1 mutant mice that had not yet developed a MPD phenotype compared to wild type mice. A somewhat higher white blood cell count was observed in Nde1 mutant mice with MPD phenotype. In the Nde1 mutant mice with or without splenomegaly we did not observe an increase in the number of B- and T-cells as measured by flowcytometer using various lymphoid cell surface markers. Based on these data we conclude that loss of Nde1 function and Nde1 haploinsufficiency results in the onset of a MPD phenotype. Earlier studies showed that Nde1 is a centrosomal protein. To test whether Nde1 is involved in spindle pole formation and chromosome segregation, we analyzed the mitotic behaviour of mouse embryo fibroblasts (MEFs). From passage 3 to passage 10, aneuploid metaphases increased much more rapidly in Nde1−/− MEFs than in the wild type controls. In addition, a broader spectrum of abnormal chromosome numbers was seen in Nde1−/− MEFs. Thus, Nde1 deficiency results in chromosomal instability. To study whether the numerical chromosome abnormalities were associated with aberrant spindle formation we stained Nde1−/− MEFs with a monoclonal antibody against tubulin. Multipolar spindles and misassembled spindles were more frequently observed in Nde1−/− MEFs compared to wild type cells. We conclude that the abnormal chromosomal segregation of Nde1−/− MEFs is associated with defective mitotic spindle structure. Since Nde1 is disrupted in the majority of inv(16) positive AML patients and the disruption in mice resulted in genetic instability and the onset of myeloproliferative disease, we suggest that the disruption of Nde1 in inv(16)+ AML may serve as additional genetic defect that contributes to leukemogenesis.

Decomposition of Continua and Prime Ends

Let f map the unit disk onto a simply connected domain G. In this paper we consider decompositions of the form \(\partial G=C_{1}\cup \cdots \cup C_{m}\), in particular the case where Cj are continua and we investigate inclusions between the cluster sets \(I(f,\zeta),I^{\pm}(f,\zeta),\Pi (f,\zeta)\) and the given sets Cj. In this context the concept of a indecomposable continuum appears in a natural way.

Application of Beneficial Microorganisms to Seeds during Drum Priming

Five microbial plant growth promoters or biocontrol agents (Pseudomonas fluorescens CHA0, Pseudomonas sp. AB842, Bacillus subtilis MBI600, Trichoderma harzianum T22 and T. virens G20) were assessed for ability to proliferate on seeds of carrot, parsnip and leek. In small-scale priming systems, both pseudomonads and MBI600 (when applied as cells) at levels between 105 and 106 cfu g−1 seed were able to colonise all seeds at the end of priming (240 h) despite initial poor recovery after addition of the cells in some cases. Pf CHA0 was a particularly aggressive seed coloniser often comprising the total pseudomonad population at the end of priming. Drying the seed after priming resulted in <1 log10 cfu g−1 seed loss for the pseudomonads but greater losses for MBI600 on carrot and leek seed. Application of spores of MBI600 resulted in little loss in cfu g−1 seed following addition of the cells and these levels were maintained throughout the priming period and after drying back. Both T22 and G20 were recovered from carrot and parsnip at the end of priming and in general reflected survival of the inoculum rather than proliferation. T22 and G20 could not be recovered from leek seed following priming. Comparisons were made between proliferation and survival in large-scale drum priming with post-priming application of microorganisms. Pf CHA0 proliferated on carrot and parsnip seed during drum priming and survived the drying back process whereas there was no recovery when applied as a post-priming treatment. In contrast, MBI600 could not be recovered when applied during priming as cells, whereas recovery was good when applied post-priming as spores. T22 spores could be applied in either manner. Post-priming application of metalaxyl and thiabendazole had variable effects on microorganism recovery. The significance of the application of microorganisms to seeds during priming is discussed.

On Prime Ends and Plane Continua

Let $f$ be a conformal map of the unit disk ${\bb D}$ onto the domain $G \subset \hat{\bb C} = {\bb C} \cup \{\infty\}$ . We shall always use the spherical metric in $\hat{\bb C}$ . Caratheodory [ 3 ] introduced the concept of a prime end of $G$ in order to describe the boundary behaviour of $f$ in geometric terms; see for example [ 6 , Chapter 9] or [ 12 , Section 2.4]. There is a bijective map $\hat{f}$ of ${\bb T} = \partial {\bb D}$ onto the set of prime ends of $G$ .

Ribosomal DNA Sequence Divergence and Group I Introns within the Leucostoma Species L. cinctum, L. persoonii, and L. parapersoonii sp. nov., Ascomycetes That Cause Cytospora Canker of Fruit Trees

Leucostoma species that are the causal agents of Cytospora canker of stone and pome fruit trees were studied in detail. DNA sequence of the internal transcribed spacer regions and the 5.8S of the nuclear ribosomal DNA operon (ITS rDNA) supplied sufficient characters to assess the phylogenetic relationships among species of Leucostoma, Valsa, Valsella, and related anamorphs in Cytospora. Parsimony analysis of the aligned sequence divided Cytospora isolates from fruit trees into clades that generally agreed with the morphological species concepts, and with some of the phenetic groupings (PG 1-6) identified previously by isozyme analysis and cultural characteristics. Phylogenetic analysis inferred that isolates of L. persoonii formed two well-resolved clades distinct from isolates of L. cinctum. Phylogenetic analysis of the ITS rDNA, isozyme analysis, and cultural characteristics supported the inference that L. persoonii groups PG 2 and PG 3 were populations of a new species apparently more genetically different from L. persoonii PG 1 than from isolates representative of L. massariana, L. niveum, L. translucens, and Valsella melastoma. The new species, L. parapersoonii, was described. A diverse collection of isolates of L. cinctum, L. persoonii, and L. parapersoonii were examined for genetic variation using restriction fragment length polymorphism (RFLP) analysis of the ITS rDNA and the five prime end of the large subunit of the rDNA (LSU rDNA). HinfI and HpaII endonucleases were each useful in dividing the Leucostoma isolates into RFLP profiles corresponding to the isozyme phenetic groups, PG 1-6. RFLP analysis was more effective than isozyme analysis in uncovering variation among isolates of L. persoonii PG 1, but less effective within L. cinctum populations. Isolates representative of seven of the L. persoonii formae speciales proposed by G. Défago in 1935 were found to be genetically diverse isolates of PG 1. Two large insertions, 415 and 309 nucleotides long, in the small subunit (SSU) of the nuclear rDNA of L. cinctum were identified as Group 1 introns; intron 1 at position 943 and intron 2 at position 1199. The two introns were found to be consistently present in isolates of L. cinctum PG 4 and PG 5 and absent from L. cinctum PG 6 isolates, despite the similarity of the ITS sequence and teleomorph morphology. Intron 1 was of subgroup 1C1 whereas intron 2 was of an unknown subgroup. RFLP patterns and presence/absence of introns were useful characters for expediting the identification of cultures of Leucostoma isolated from stone and pome fruit cankers. RFLP patterns from 13 endonucleases provided an effective method for selecting an array of diverse PG 1 isolates useful in screening plant germplasm for disease-resistance.

Ribosomal DNA sequence divergence and group I introns within the Leucostoma species L. cinctum, L. persoonii, and L. parapersoonii sp. nov., ascomycetes that cause Cytospora canker of fruit trees

Leucostoma species that are the causal agents of Cytospora canker of stone and pome fruit trees were studied in detail. DNA sequence of the internal transcribed spacer regions and the 5.8S of the nuclear ribosomal DNA operon (ITS rDNA) supplied sufficient characters to assess the phylogenetic relationships among species of Leucostoma, Valsa, Valsella, and related anamorphs in Cytospora. Parsimony analysis of the aligned sequence divided Cytospora isolates from fruit trees into clades that generally agreed with the morphological species concepts, and with some of the phenetic groupings (PG 1–6) identified previously by isozyme analysis and cultural characteristics. Phylogenetic analysis inferred that isolates of L. persoonii formed two well-resolved clades distinct from isolates of L. cinctum. Phylogenetic analysis of the ITS rDNA, isozyme analysis, and cultural characteristics supported the inference that L. persoonii groups PG 2 and PG 3 were populations of a new species apparently more genetically different from L. persoonii PG 1 than from isolates representative of L. massariana, L. niveum, L. translucens, and Valsella melastoma. The new species, L. parapersoonii, was described. A diverse collection of isolates of L. cinctum, L. persoonii, and L. parapersoonii were examined for genetic variation using restriction fragment length polymorphism (RFLP) analysis of the ITS rDNA and the five prime end of the large subunit of the rDNA (LSU rDNA). HinfI and HpaII endonucleases were each useful in dividing the Leucostoma isolates into RFLP profiles corresponding to the isozyme phenetic groups, PG 1–6. RFLP analysis was more effective than isozyme analysis in uncovering variation among isolates of L. persoonii PG 1, but less effective within L. cinctum populations. Isolates representative of seven of the L. persoonii formae speciales proposed by G. Défago in 1935 were found to be genetically diverse isolates of PG 1. Two large insertions, 415 and 309 nucleotides long, in the small subunit (SSU) of the nuclear rDNA of L. cinctum were identified as Group 1 introns; intron 1 at position 943 and intron 2 at position 1199. The two introns were found to be consistently present in isolates of L. cinctum PG 4 and PG 5 and absent from L. cinctum PG 6 isolates, despite the similarity of the ITS sequence and teleomorph morphology. Intron 1 was of subgroup 1C1 whereas intron 2 was of an unknown subgroup. RFLP patterns and presence/absence of introns were useful characters for expediting the identification of cultures of Leucostoma isolated from stone and pome fruit cankers. RFLP patterns from 13 endonucleases provided an effective method for selecting an array of diverse PG 1 isolates useful in screening plant germplasm for disease-resistance.

The region of the HSV-1 latency-associated transcript required for epinephrine-induced reactivation in the rabbit does not include the 2.0-kb intron.

Previous studies have localized the region of the latency-associated transcript (LAT) of HSV-1 responsible for epinephrine-induced reactivation in the rabbit eye model to the first 1.5 kb of the primary transcript. This region extends from the 5prime prime or minute exon of the primary LAT transcript through the 5prime prime or minute half of the LAT 2.0-kb intron. To determine whether the 5prime prime or minute end of the LAT intron contributes to the induced reactivation phenotype, three recombinant viruses containing deletions within this portion of the LAT intron were constructed. The three recombinants, containing deletions spanning a combined region of 969 bp at the 5prime prime or minute end of the LAT intron, reactivated with the wild-type frequency of 17syn+. These results indicate that the elements governing induced reactivation reside within the first 699 bp of the primary LAT transcript encoding the 5prime prime or minute LAT exon.

The 3prime prime or minute-terminal structure required for replication of Barley yellow dwarf virus RNA contains an embedded 3prime prime or minute end.

We determined the 3prime prime or minute-terminal primary and secondary structures required for replication of Barley yellow dwarf virus (BYDV) RNA in oat protoplasts. Computer predictions, nuclease probing, phylogenetic comparisons, and replication assays of specific mutants and chimeras revealed that the 3prime prime or minute-terminal 109 nucleotides (nt) form a structure with three to four stem-loops followed by a coaxially stacked helix incorporating the last four nt [(A/U)CCC]. Sequences upstream of the 109-nt region also contributed to RNA accumulation. The base-pairing but not the sequences or bulges in the stems were essential for replication, but any changes to the 3prime prime or minute-terminal helix destroyed replication. The two 3prime prime or minute-proximal tetraloops tolerated all changes, but the two 3prime prime or minute-distal tetraloops gave most efficient replication if they fit the GNRA consensus. A mutant lacking the 3prime prime or minute-proximal stem-loop produced elevated levels of less-than-full-length minus strands, and no (+) strand. We propose that a "pocket" structure is the origin of (minus sign)-strand synthesis, which is negatively regulated by the inaccessible conformation of the 3prime prime or minute terminus, thus favoring a high (+)/(minus sign) ratio. This 3prime prime or minute structure and the polymerase homologies suggest that genus Luteovirus is more closely related to the Tombusviridae family than to other Luteoviridae genera.

Completing the conformal boundary of a simply connected Lorentz surface

This paper extends Kulkarni's conformal boundary ∂L for a simply connected Lorentz surface £ to a compact conformal boundary ∂ c L. The procedure used is analogous to Caratheodory's construction (in the definite metric setting) of prime ends from the accessible points of a bounded simply connected planar domain. The space ∂ c L of conformal boundary elements is homeomorphic to the circle, and contains Kulkarni's conformal boundary ∂L as a dense subspace.

CHARACTERIZATION OF EXPRESSED SEQUENCE TAGS GENERATED FROM SKIN CDNA CLONES OF Equus caballus BY SINGLE PASS SEQUENCING

A cDNA library was built using RNA extracted from the skin tissue of an adult horse. The library was primed with oligo (dT) and sequences were directionally inserted in order to produce an expression library. The library has 5.8X 105 plaque forming units with 99.6% recombinant phage. The average insert size is 1.3 Kbp. Three hundred and thirteen expressed sequence tags (ESTs) were generated from sequencing of the 5 prime end of randomly selected skin cDNA clones. The ESTs were sequenced on an ABI 377 using Big-Dye chemistry. A similarity search was performed on each EST using the NCBI non-redundant protein database and 206 ESTs were putatively identified. Twenty six percent of the identified ESTs were redundant. The ESTs were categorized by function. The most frequently identified functional class was translational proteins.

Expression profiling of single cells using 3 prime end amplification (TPEA) PCR.

The ability to relate the physiological status of individual cells to the complement of genes they express is limited by current methodological approaches for performing these analyses. We report here the development of a robust and reproducible method for amplifying 3' sequences of mRNA derived from single cells and demonstrate that the amplified cDNA, derived from individual human lymphoblastoma cells, can be used for the expression profiling of up to 40 different genes per cell. In addition, we show that 3 prime end amplification (TPEA) PCR can be used to enable the detection of both high and low abundance mRNA species in samples harvested from live neurons in rat brain slices. This procedure will facilitate the study of complex tissue function at the cellular level.

The Pufferfish Gene Map.

The earliest true vertebrates are represented by fish. The largest fish class, the teleosts or bony fish, contain nearly 20,000 known species. Although this class comprises only about half of all the known vertebrate species, they are probably the least well studied at the molecular level among vertebrate phyla. Until studies of the mid-1990s on the zebrafish {Danio rerio) and the pufferfish (Fugu rubripes) (2 very different teleosts), only a handful of fish genes have been listed in databases. However, because of the evolutionary position of fish, many important aspects of molecular evolution may be addressed by their study. The genomic organization of fish is very different from that of mammals. Fish chromosomes on the whole are much smaller than those of mammals. The way in which the chromosomes are constructed has a different organization. For example, fish chromosomes do not show Giemsa banding, which has been attributed to the fact that they have a less welldefined isochore structure, that is, they do not posses guanine and cytosine-poor and guanine and cytosine-rich regions (Medrano and others 1988). They also have a higher frequency of the dinucleotide CpG (Elgar 1996), the suppression of which is associated with the 5 prime ends of genes in mammals. This article and others in this Journal issue address whether such differences in the organization of other species' genes along respective chromosomes bears any resemblance to mammalian gene orders. Pufferfish are members of the Tetraodontiformes, denoting 4 teeth. The approximately 200 to 300 species in the order are found in freshwater, brackish, and marine environments. F. rubripes (also called ToraFugu or TakiFugu) (Figure 1) are marine fish found in the temperate coastal waters of Japan. Because of their size and growth rate (potentially 60 cm long and weighing more than 2 kg), they are farmed commercially and eaten as a delicacy. Since they are known to be aggressive, however, they require a large marine tank. Because they do not mature sexually for 2 to 3 yr, they are difficult and unsuitable for breeding. F. rubripes, like many of the other 23 mem-

A Noncleavable Retro-Binding Peptide That Spans the Substrate Binding Cleft of Serine Proteases. Atomic Structure of Nazumamide A: Human Thrombin

The 2.0 A resolution crystal structure of the retro-binding natural product nazumamide A (NAZA) complexed with human thrombin is presented. The crystal structure shows that the retro-binding nazumamide A is noncleavable and extends into the prime end of the substrate binding cleft. The data suggest ideas for SAR and combinatorial modification of nazumamide A to create a second generation of nazumamide-like compounds which are potent and specific for human thrombin. This crystal structure indicates that fresh ideas for the generation of novel and specific inhibitors may arise from examining weak binding compounds. Additionally, the crystal structure demonstrates the utility of crystallographic analysis of natural product:protein complexes.

Indecomposable Continua and the Julia Sets of Polynomials

We find several necessary and sufficient conditions for the Julia set $J$ of a polynomial of degree $d \geqslant 2$ to be an indecomposable continuum. One condition that may be easier to check than others is the following: Suppose $J$ is connected; then $J$ is an indecomposable continuum iff the impression of some prime end of the unbounded complementary domain of $J$ has interior in $J$.

Expression of a common cellular phospholipase A2 by human intrauterine tissues

A subclone of the three prime (3′) end of a human extrapancreatic phospholipase cDNA has been generated using the polymerase chain reaction. This has been used to detect mRNA transcripts in RNA extracted from human amnion, chorion-decidua, placenta and myometrium. The carboxyl-terminus of phospholipases A2 is poorly conserved but hybridisation remained stable under conditions of high stringency. This supports the hypothesis that these tissues express a common cellular phospholipase A2 which is identical to that expressed in platelets.

Indecomposable continua and the Julia sets of polynomials

We find several necessary and sufficient conditions for the Julia set J J of a polynomial of degree d ⩾ 2 d \geqslant 2 to be an indecomposable continuum. One condition that may be easier to check than others is the following: Suppose J J is connected; then J J is an indecomposable continuum iff the impression of some prime end of the unbounded complementary domain of J J has interior in J J .

A fixed point theorem for plane separating continua

If an orientation preserving plane homeomorphism F has a fixed prime end associated with an invariant continuum Δ that separates the plane into exactly two domains, then F has a fixed point in Δ.

Protein synthesis during osmopriming of leek (Allium porrum L.) seeds

AbstractAn osmotic priming treatment of 14 days in a −1.0 MPa polyethylene glycol solution improves the germination performance of a highvigour seed lot of leek (Allium porrum L., cv. Verina). Using in vivo pulse-labelling, two-dimensional polyacrylamide gel electrophoresis and fluorography, five polypeptides were found to be synthesized in embryonic tissue at 14 days of priming that were not present at 4 days of germination without prior priming. These polypeptides were still synthesized at 6 h of germination following priming. This time point lies in the 6–12 h lag phase in protein synthesis previously observed in leek embryo tissue during germination of primed seeds where there is little increase in the rate of synthesis over that seen at the end of priming. None of the polypeptides was synthesized at 2 days of germination after priming, a period of seedling growth. The five polypeptides appear to be specifically associated with the priming period. Two additional polypeptides were found in leek embryos that were synthesized at higher levels at the end of priming than during germination alone. These continued to be synthesized, at lower levels in leek embryo tissue upon germination after priming. Several polypeptides were identified in leek endosperm tissue which were synthesized at higher levels during priming than during germination and also two polypeptides whose synthesis appeared to be specific to germination.

Periodicity and Decomposability of Basin Boundaries with Irrational Maps on Prime Ends

Periodicity and Decomposability of Basin Boundaries with Irrational Maps on Prime Ends