bluuebunny/crossref_metadata_2025 huggingface dataset:
Cisplatin-DNA adducts are molecular decoys for the ribosomal RNA transcription factor hUBF (human upstream binding factor).
Authors: Treiber D K , Zhai X , Jantzen H M , Essigmann J M
Published: May 2006

The toxicity of DNA-damaging agents is widely believed to result from the formation of lesions that block polymerases or disrupt the integrity of the genome. A mechanism heretofore not addressed is that DNA damage may titrate essential DNA-binding proteins away from their natural sites of action. This report shows that the ribosomal RNA (rRNA) transcription factor hUBF (human upstream binding factor) binds with striking affinity (Kd(app) approximately 60 pM) to the intrastrand cis-[Pt(NH3)2](2+-d(GpG) crosslink formed by the anticancer drug cis-diamminedichloroplatinum(II) (cisplatin). When protein blots of human cell extracts are probed with cisplatin-modified DNA, 97- and 94-kDa proteins are detected, consistent with the known sites of hUBF species. A similar analysis of blots containing in vitro translated hUBF confirmed that the protein binds cisplatin adducts with high specificity. By contrast, DNA adducts of the clinically ineffective trans isomer of cisplatin, trans-diamminedichloroplatinum(II), are not recognized by hUBF. DNase I inhibition patterns of hUBF bound to a 100-base-pair DNA fragment containing a centrally located cis-[Pt(NH3)2](2+)-d(GpG) crosslink reveal specific protein-DNA interactions in a 14-base-pair region flanking the adduct. The affinity of hUBF for the rRNA promoter is similar (Kd(app) approximately 18 pM) to that measured for the cisplatin adduct. In addition, we observe that the hUBF-promoter interaction is highly sensitive to the antagonistic effects of cisplatin-DNA adducts. These results suggest that a cisplatin-mediated transcription-factor-hijacking mechanisms could disrupt rRNA synthesis, which is stimulated in proliferating cells.
Regulation of competence-specific gene expression by Mec-mediated protein-protein interaction in Bacillus subtilis.
Authors: Kong L , Dubnau D
Published: May 2006

The expression of competence genes in Bacillus subtilis is controlled by a signal transduction cascade which increases the expression of a competence transcription factor (CTF, encoded by comK) during the transition from exponential growth to stationary phase. The transcription of CTF (ComK) is decreased by the product of the mecA gene, and this inhibition is relieved in response to an unknown signal received from upstream in the regulatory pathway. Inactivation of either mecA or another gene, mecB, results in overproduction of ComK. We show here that the concentration of MecA protein does not vary markedly with culture medium, as a function of growth stage, or in competent and noncompetent cells. We also show that MecA can interact directly with ComK. Finally, evidence is presented suggesting that MecB functions prior to MecA in the signaling pathway. A model is discussed which involves the sequestration of ComK by MecA binding and the release of the transcription factor when an appropriate signal is relayed to MecA by MecB.
Characterization of nucleoside-diphosphate kinase from Pseudomonas aeruginosa: complex formation with succinyl-CoA synthetase.
Authors: Kavanaugh-Black A , Connolly D M , Chugani S A , Chakrabarty A M
Published: May 2006

The enzyme nucleoside-diphosphate kinase (Ndk), responsible for the conversion of (deoxy)ribonucleoside diphosphates to their corresponding triphosphates, has been purified from Pseudomonas aeruginosa. The N-terminal 12 amino acid sequence of P. aeruginosa Ndk shows significant homology with that of Myxococcus xanthus and that of Escherichia coli. Ndk enzyme activity is also associated with succinyl-CoA synthetase activity in P. aeruginosa, whose alpha and beta subunits show extensive sequence homology with those of E. coli and Dictyostelium discoideum. The 33-kDa alpha subunit of succinyl-CoA synthetase of P. aeruginosa appears to undergo autophosphorylation in the presence of either ATP or GTP, although the presence of small amounts of Ndk activity may influence the level of such phosphorylation.
Induction of tyrosine hydroxylase gene expression by a nonneuronal nonpituitary-mediated mechanism in immobilization stress.
Authors: Nankova B , Kvetnanský R , McMahon A , Viskupic E , Hiremagalur B , Frankle G , Fukuhara K , Kopin I J , Sabban E L
Published: May 2006

Stress stimulates the sympathoadrenal system, causing activation of the catecholamine biosynthetic enzymes. Here we examine the changes of gene expression of tyrosine hydroxylase (TH; EC 1.14.16.2), the initial enzyme of catecholamine biosynthesis, with stress. A single immobilization of rats led to a large transient elevation in TH mRNA and a small elevation in TH immunoreactive protein and activity. Repeated daily immobilizations triggered more sustained changes in TH mRNA levels. After two immobilizations, the levels remained elevated even 3 days later. The rise in TH mRNA was followed by increased immunoreactive protein but only a small elevation in activity. With seven repeated immobilizations, the animals did not appear to adapt and still manifested a further rise in TH mRNA. TH activity was markedly elevated and returned to control levels 7 days after the immobilization. The rise in TH mRNA with a single immobilization occurred even in adrenals of hypophysectomized rats that underwent splanchnic nerve section. Immobilization for 30 min was sufficient to increase TH mRNA. The effect was abolished by the transcriptional inhibitor actinomycin D. Mobility gel-shift assays revealed increased binding of c-Fos and c-Jun to the AP-1 transcription factor site after a single immobilization, and the binding was not further elevated with repeated stress. This study shows that a single immobilization can activate TH gene expression by a nonneuronal nonpituitary-mediated pathway associated with increased binding of AP-1 transcription factors.
Decomposition of a mixture of signals in a model of the olfactory bulb.
Authors: Hendin O , Horn D , Hopfield J J
Published: May 2006

We describe models for the olfactory bulb which perform separation and decomposition of mixed odor inputs from different sources. The odors are unknown to the system; hence this is an analog and extension of the engineering problem of blind separation of signals. The separation process makes use of the different temporal fluctuations of the input odors which occur under natural conditions. We discuss two possibilities, one relying on a specific architecture connecting modules with the same sensory inputs and the other assuming that the modules (e.g., glomeruli) have different receptive fields in odor space. We compare the implications of these models for the testing of mixed odors from a single source.
Close linkage to chromosome 3p and conservation of ancestral founding haplotype in hereditary nonpolyposis colorectal cancer families.
Authors: Nyström-Lahti M , Sistonen P , Mecklin J P , Pylkkänen L , Aaltonen L A , Järvinen H , Weissenbach J , de la Chapelle A , Peltomäki P
Published: May 2006

A susceptibility to hereditary nonpolyposis colorectal cancer (HNPCC) was recently shown to be due to mutations in the MSH2 gene on chromosome 2p. A second susceptibility locus has been mapped to chromosome 3p in two families. The present report describes the results of a genetic study of Finnish HNPCC kindreds. Of 18 apparently unrelated families living in different parts of the country, 11 could be genealogically traced to a common ancestry dating at least 13 generations back in a small geographic area. Linkage studies were possible in 9 families, revealing conclusive or probable linkage to markers on 3p in 8. Five of these were among those having shared ancestry. The location of the gene was refined by a linkage study comprising 12 marker loci. By analysis of recombinations in such families, the HNPCC locus could be assigned to the 1-centimorgan interval between marker loci D3S1561 and D3S1298. A haplotype encompassing 10 centimorgans around the HNPCC locus was conserved in five of the pedigrees with shared ancestry and present in 2 further families in which linkage analysis was not possible. Our results suggest the presence of a widespread single ancestral founding mutation. Moreover, the map position of the 3p gene for HNPCC susceptibility was greatly refined.
Production of calves by transfer of nuclei from cultured inner cell mass cells.
Authors: Sims M , First N L
Published: May 2006

We report here the isolation and in vitro culture of bovine inner cell mass (ICM) cells and the use of ICM cells in nuclear transfer to produce totipotent blastocysts that resulted in calves born. Of 15 cell lines represented in this study, 13 were derived from immunosurgically isolated ICM of 3 in vitro produced day 9-10 bovine blastocysts, while 2 lines were derived from single blastocysts. Approximately 70% of attempted cell lines became established cell lines when started from 3 ICMs. The ability to establish cell lines was dependent on the number of ICMs starting the line. Sire differences were noted in the ability of ICMs to establish cell lines and to form blastocysts. The cell lines were cultured as a low cell density suspension in the medium CR1aa plus selenium, insulin, and transferrin (SIT) and 5% fetal calf serum (FCS) for 6-101 days before use in nuclear transfer, at which time some had multiplied to more than 2000 cells. If allowed to aggregate, cells of established cell lines formed embryoid bodies. A total of 659 nuclear transfer clones were made by fusing the ES cells into enucleated oocytes with polyethylene glycol; 460 of these fused, based on cleavage (70%). After culture of the clones for 7 days in vitro in CR1aa/SIT/5% FCS, 109 (24%) of those fused became blastocysts. Thirty-four blastocysts were transferred into uteri of 27 cows, and 13 cows (49%) became pregnant. Four of the 13 cows gave birth to 4 normal calves. DNA typing showed the calves to be derived from the respective sires of the cell lines. The calves were derived from cultures of less than 28 days.
Rch1, a protein that specifically interacts with the RAG-1 recombination-activating protein.
Authors: Cuomo C A , Kirch S A , Gyuris J , Brent R , Oettinger M A
Published: May 2006

RAG1 and RAG2 are lymphoid-specific genes that together induce V(D)J recombinase activity in a variety of nonlymphoid cell types. While no other lymphoid-specific factors are required to induce recombination, other factors with more widespread expression patterns have been implicated in the reaction. However, none of these factors have been cloned, and their relationship to the RAG proteins is unclear. Using the yeast two-hybrid assay, we have identified RCH1, a gene encoding a protein of molecular weight 58,000 that interacts specifically with RAG-1. The predicted Rch1 protein sequence is 47% identical to yeast SRP1, a protein associated with the nuclear envelope. A truncated form of Rch1, which retains the ability to interact with RAG-1, reduces V(D)J recombination activity in HeLa cells.
Mevalonate-mediated suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase function in alpha-toxin-perforated cells.
Authors: Giron M D , Havel C M , Watson J A
Published: May 2006

The regulation of mevalonic acid synthesis requires both nonsterol isopentenoid and sterol regulatory signal molecules. A primary target of this multivalent control process is the enzyme which catalyzes mevalonate synthesis: 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (EC 1.1.1.34). In this report Staphylococcus aureus alpha-toxin perforated Chinese hamster ovary cells were used to facilitate the identification of isopentenoidogenic reactions and metabolites required for mevalonate-mediated loss of HMG-CoA reductase activity. alpha-Toxin-perforated cells retained the capacity to decrease, upon demand, HMG-CoA reductase activity and protein in response to mevalonate or isopentenoid pyrophosphate esters. Also, it was deduced with highly specific metabolic inhibitors, that conversion of farnesyl 1-diphosphate to squalene was required for mevalonate-mediated suppression of reductase activity. Since squalene (2 microM) did not downregulate reductase activity, pre-squalene pyrophosphate or a derivative, or polyprenyl-1-pyrophosphate-generated inorganic pyrophosphate, or a combination of these metabolites are proposed as candidate regulatory nonsterol isopentenoid signal molecules.
Host cell responses to Listeria monocytogenes infection include differential transcription of host stress genes involved in signal transduction.
Authors: Schwan W R , Goebel W
Published: May 2006

We examined the effect of Listeria monocytogenes infection of J774 macrophage-like mouse cells on induction of several stress genes, including genes for heat shock proteins (HSPs) and a protein-tyrosine phosphatase (PTP), to understand the host response in various steps of the bacterial invasion process. Exposure to wild-type L. monocytogenes strain EGD elicited an early induction of HSP70 mRNA with a corresponding early appearance of HSP70 protein. Cytochalasin D pretreatment prevented the induction of HSP70 mRNA in L. monocytogenes-infected macrophages. After a 2-hr infection with L. monocytogenes, PTP and to a lesser extent HSP90 mRNA levels were elevated. A listeriolysin-negative mutant of L. monocytogenes strain EGD and a noninvasive species of Listeria, Listeria innocua, did not induce PTP or HSP90 mRNA in infected macrophages. Mutations in other virulence genes did not affect transcription of PTP or HSP90. Expression of HSP60 mRNA remained constant over the time course studied in wild-type or mutant strains. These results suggest that phagocytosis of L. monocytogenes triggers transcription of HSP70 mRNA in macrophages; however, escape from the phagosome appears to be necessary for induction of PTP and HSP90 mRNA. Since both PTP and HSP90 may have links with signal transduction pathways in eukaryotic cells, the induction of these mRNAs suggests a role for L. monocytogenes in influencing the signal transduction routes of the host cell.
Identification of a human melanoma antigen recognized by tumor-infiltrating lymphocytes associated with in vivo tumor rejection.
Authors: Kawakami Y , Eliyahu S , Delgado C H , Robbins P F , Sakaguchi K , Appella E , Yannelli J R , Adema G J , Miki T , Rosenberg S A
Published: May 2006

The cultured T-cell line TIL1200, established from the tumor-infiltrating lymphocytes (TILs) of a patient with advanced metastatic melanoma, recognized an antigen on most HLA-A2+ melanomas and on all HLA-A2+ cultured neonatal melanocytes in an HLA-A2 restricted manner but not on other types of tissues or cell lines tested. A cDNA encoding an antigen recognized by TIL1200 was isolated by screening an HLA-A2+ breast cancer cell line transfected with an expression cDNA library prepared from an HLA-A2+ melanoma cell line. The nucleotide and amino acid sequences of this cDNA were almost identical to the genes encoding glycoprotein gp100 or Pmel17 previously registered in the GenBank. Expression of this gene was restricted to melanoma and melanocyte cell lines and retina but was not expressed on other fresh or cultured normal tissues or other types of tumor tested. The cell line transfected with this cDNA also expressed antigen recognized by the melanoma-specific antibody HMB45 that bound to gp100. A synthetic 10-amino acid peptide derived from gp100 was recognized by TIL1200 in the context of HLA-A2.1. Since the administration of TIL1200 plus interleukin 2 resulted in regression of metastatic cancer in the autologous patient, gp100 is a possible tumor rejection antigen and may be useful for the development of immunotherapies for patients with melanoma.
Altered natural killer cell repertoire in Tap-1 mutant mice.
Authors: Ljunggren H G , Van Kaer L , Ploegh H L , Tonegawa S
Published: May 2006

We have analyzed the specificity and function of natural killer (NK) cells in mice with a homozygous deletion of the major histocompatibility complex (MHC)-encoded transporter gene associated with MHC class I-restricted antigen presentation (Tap-1). These mice express very low levels of class I molecules at the cell surface, and these molecules are either devoid of peptide or occupied only by TAP-independent peptides. NK cells in Tap-1 -/- mice, through normal in number, appeared tolerant toward autologous Tap-1 -/- Con A-activated blasts, Tap-1 -/- as well as allogeneic BALB/c bone marrow cells, and RMA-S tumor cell grafts. In contrast, they killed YAC-1 cells as efficiently as did NK cells from wild-type mice. Defective Tap-1 expression was sufficient to render nontransformed target cells sensitive to NK cell-mediated lysis. It is concluded that proper expression of TAP molecules is necessary for normal development of NK cells, as well as for rendering target cells resistant to NK cell-mediated lysis. These results support the hypothesis that class I molecules of the MHC influence the sensitivity of target cells to lysis by NK cells, as well as the development of the NK cell repertoire.
Disruption of hydrogen bonding between plant cell wall polymers by proteins that induce wall extension.
Authors: McQueen-Mason S , Cosgrove D J
Published: May 2006

Plant cell enlargement is controlled by the ability of the constraining cell wall to expand. This ability has been postulated to be under the control of polysaccharide hydrolases or transferases that weaken or rearrange the loadbearing polymeric networks in the wall. We recently identified a family of wall proteins, called expansins, that catalyze the extension of isolated plant cell walls. Here we report that these proteins mechanically weaken pure cellulose paper in extension assays and stress relaxation assays, without detectable cellulase activity (exo- or endo- type). Because paper derives its mechanical strength from hydrogen bonding between cellulose microfibrils, we conclude that expansins can disrupt hydrogen bonding between cellulose fibers. This conclusion is further supported by experiments in which expansin-mediated wall extension (i) was increased by 2 M urea (which should weaken hydrogen bonding between wall polymers) and (ii) was decreased by replacement of water with deuterated water, which has a stronger hydrogen bond. The temperature sensitivity of expansin-mediated wall extension suggests that units of 3 or 4 hydrogen bonds are broken by the action of expansins. In the growing cell wall, expansin action is likely to catalyze slippage between cellulose microfibrils and the polysaccharide matrix, and thereby catalyze wall stress relaxation, followed by wall surface expansion and plant cell enlargement.
Structure, function, and evolution of mouse TL genes, nonclassical class I genes of the major histocompatibility complex.
Authors: Obata Y , Satta Y , Moriwaki K , Shiroishi T , Hasegawa H , Takahashi T , Takahata N
Published: May 2006

In contrast to well-studied "classical" class I genes of the major histocompatibility complex (MHC), the biology of nonclassical class I genes remains largely unexamined. The mouse TL genes constitute one of the best defined systems among nonclassical class I genes in the T region of the MHC. To elucidate the function and the evolution of TL genes and their relationship to classical class I genes, seven TL DNA sequences, including one from a Japanese wild mouse, were examined and compared with those of several mouse and human classical class I genes. The TL genes differ from either classical class I genes or pseudogenes in the extent and pattern of nucleotide substitutions. Natural selection appears to have operated so as to preserve the function of TL, which might have been acquired in an early stage of its evolution. In a putative peptide-binding region encoded by TL genes, the rate of nonsynonymous (amino acid replacing) substitution is considerably lower than that of synonymous substitution. This conservation is completely opposite that in classical class I genes, in which the peptide-binding region has evolved to diversify amino acid sequences so as to recognize a variety of antigens. Thus, it is suggested that the function of TL antigens is distinct from that of classical class I antigens and is related to the recognition of a relatively restricted repertoire of antigens and their presentation to T-cell receptors.
The Fenton oxidation mechanism: reactivities of biologically relevant substrates with two oxidizing intermediates differ from those predicted for the hydroxyl radical.
Authors: Wink D A , Nims R W , Saavedra J E , Utermahlen W E , Ford P C
Published: May 2006

The application of kinetic probes that allow one to determine relative reactivities of biologically relevant substrates with oxidizing intermediates in the Fenton reagent (H2O2 plus Fe2+ in acidic aqueous solution) is described. These results lead to the conclusion that there are two key intermediates with very different reactivity patterns. One (X) is proposed to be an iron complex formed via direct reaction of H2O2 with Fe2+, which reacts with N-nitrosodimethylamine to generate a strong transient absorption at 450 nm. This provides a sensitive spectrophotometric probe of the competitive reactivities toward X of biologically relevant substrates such as nucleic acid components and amino acids. The second intermediate (Y) is probed by its oxidation of the Ru(bpy)2+3 ion (bpy = 2,2'-bipyridine) to a product with an absorption band centered at 500 nm. In the absence of other substrates, Ru(bpy)2+3 is oxidized at rates independent of the Ru concentration, but the product yield is diminished by competing reactions with substrates that can intercept X. Competition studies demonstrate reactivity patterns for X and Y that are clearly distinct from the pattern predicted for the hydroxyl radical, the intermediate commonly invoked in discussions of Fenton oxidations. These data require reevaluation of the mechanisms by which the Fenton reagent oxidizes biological substrates.
Experimental control of neoplastic progression in cell populations: Foulds' rules revisited.
Authors: Rubin H
Published: May 2006

Foulds introduced six rules of tumor progression based on his observations of spontaneous mammary cancer in mice and generalized them to all forms of neoplasia [Foulds, L. (1954) Cancer Res. 14, 327-339 and Foulds, L. (1969) Neoplastic Development (Academic, New York), Vol. 1, preface and pp. 72-74.] Rules III, IV, and V are considered controversial, and research in animals seems inadequate to resolve the controversies. A subline of NIH 3T3 cells undergoes progressive transformation to produce foci of increasing population density when repeatedly constrained by sequential rounds of growth to and maintenance at confluence. Analysis of the results provides a cellular basis for rules III, IV, and V. Rule III states that progression is independent of the growth of the tumor and occurs in tumors that are arrested. Cell culture shows that progression is actually favored by constraint of growth, a result inconsistent with a major role for point mutations in progression. Indeed, there is a suggestion that the transformation may arise from chromatin changes preceding apoptosis. Rule IV states that progression can be gradual or abrupt but the latter conclusion has been frequently criticized. Cell culture exhibits both forms of progression but, in particular, eliminates the doubt about the abrupt form. Rule V, which is in a sense an extension of rule IV, states that progression follows one of alternative paths of development. The results in culture indicate that every independent transforming event gives rise to foci of unique morphology. Thus, even for the single characteristic of transformed focus morphology, many alternative paths to neoplasia are available to cells. In addition to clarifying the rules of progression, a method is described for pinpointing the time of the occurrence of events that are only expressed as dense foci after a variable lag time. The results in culture reinforce Foulds' conclusion that neoplastic development is primarily an epigenetically driven process and identify some of the cellular interactions that underlie that process.
Disparate rates, differing fates: tempo and mode of evolution changed from the Precambrian to the Phanerozoic.
Authors: Schopf J W
Published: May 2006

Over the past quarter century, detailed genus- and species-level similarities in cellular morphology between described taxa of Precambrian microfossils and extant cyanobacteria have been noted and regarded as biologically and taxonomically significant by numerous workers world-wide. Such similarities are particularly well documented for members of the Oscillatoriaceae and Chroococcaceae, the two most abundant and widespread Precambrian cyanobacterial families. For species of two additional families, the Entophysalidaceae and Pleurocapsaceae, species-level morphologic similarities are supported by in-depth fossil-modern comparisons of environment, taphonomy, development, and behavior. Morphologically and probably physiologically as well, such cyanobacterial "living fossils" have exhibited an extraordinarily slow (hypobradytelic) rate of evolutionary change, evidently a result of the broad ecologic tolerance characteristic of many members of the group and a striking example of G. G. Simpson's [Simpson, G.G. (1944) Tempo and Mode in Evolution (Columbia Univ. Press, New York)] "rule of the survival of the relatively unspecialized." In both tempo and mode of evolution, much of the Precambrian history of life--that dominated by microscopic cyanobacteria and related prokaryotes--appears to have differed markedly from the more recent Phanerozoic evolution megascopic, horotelic, adaptationally specialized eukaryotes.
Late Precambrian bilaterians: grades and clades.
Authors: Valentine J W
Published: May 2006

A broad variety of body plans and subplans appear during a period of perhaps 8 million years (my) within the Early Cambrian, an unequaled explosion of morphological novelty, the ancestral lineages represented chiefly or entirely by trace fossils. Evidence from the fossil record can be combined with that from molecular phylogenetic trees to suggest that the last common ancestor of (i) protostomes and deuterostomes was a roundish worm with a blood vascular system and (ii) of arthropods and annelids was similar, with a hydrostatic hemocoel; these forms are probably among trace makers of the late Precambrian. Cell-phenotype numbers in living phyla, and a model of cell-phenotype number increase, suggest an origin of metazoans near 600 my ago, followed by a passive rise in body-plan complexity. Living phyla appearing during the Cambrian explosion have a Hox/HOM gene cluster, implying its presence in the common ancestral trace makers. The explosion required a repatterning of gene expression that mediated the development of novel body plans but evidently did not require an important, abrupt increase in genomic or morphologic complexity.
Participation of cyclin A in Myc-induced apoptosis.
Authors: Hoang A T , Cohen K J , Barrett J F , Bergstrom D A , Dang C V
Published: May 2006

The involvement of c-Myc in cellular proliferation or apoptosis has been linked to differential cyclin gene expression. We observed that in both proliferating cells and cells undergoing apoptosis, cyclin A (but not B, C, D1, and E) mRNA level was elevated in unsynchronized Myc-overexpressing cells when compared with parental Rat1a fibroblasts. We further demonstrated that Zn(2+)-inducible cyclin A expression was sufficient to cause apoptosis. When Myc-induced apoptosis was blocked by coexpression of Bcl-2, the levels of cyclin C, D1, and E mRNAs were also elevated. Thus, while apoptosis induced by c-Myc is associated with an elevated cyclin A mRNA level, protection from apoptosis by coexpressed Bcl-2 is associated with a complementary increase in cyclin C, D1, and E mRNAs.
Yeast Srp1p has homology to armadillo/plakoglobin/beta-catenin and participates in apparently multiple nuclear functions including the maintenance of the nucleolar structure.
Authors: Yano R , Oakes M L , Tabb M M , Nomura M
Published: May 2006

SRP1, a suppressor of certain temperature-sensitive mutations in RNA polymerase I in Saccharomyces cerevisiae, encodes a protein that is associated with nuclear pores. By using a system of conditional SRP1 expression and by isolating temperature-sensitive srp1 mutants, we have demonstrated that Srp1p is essential for maintenance of the crescent-shaped nucleolar structure, RNA transcription, and the proper functions of microtubules as inferred from analysis of nuclear division/segregation and immunofluorescence microscopy of microtubules. Different mutant alleles showed significantly different phenotypes in relation to these apparently multiple functional roles of the protein. We have also found that eight imperfect 42-amino-acid tandem repeats present in Srp1p are similar to the 42-amino-acid repeats in armadillo/plakoglobin/beta-catenin proteins present in adhesive junction complexes of higher eukaryotes. We discuss this similarity in connection with the observed pleiotropic effects of srp1 mutations.
Expression of wild-type and mutant simian virus 40 large tumor antigens in villus-associated enterocytes of transgenic mice.
Authors: Kim S H , Roth K A , Coopersmith C M , Pipas J M , Gordon J I
Published: May 2006

The four principal gut epithelial cell lineages undergo continuous and rapid renewal during a geographically well-organized migration along the crypt-to-villus axis. The molecules that regulate their proliferation and differentiation programs are largely unknown. The large tumor antigen (TAg) of wild-type (wt) simian virus 40 (SV40) and its mutant derivatives represent tools for describing the contributions of regulators of the cell cycle to the proliferative state of each lineage. Expression of SV40 TAgwt in postmitotic, villus-associated enterocytes of transgenic mice causes them to reenter the cell cycle without an apparent effect on their state of differentiation. When human KRAS with a Val-12 substitution ([Val12]KRAS) is coexpressed with SV40 TAgwt in villus enterocytes of bitransgenic animals, the two oncoproteins cooperate to produce dedifferentiation (dysplasia). SV40 mutant d11137 expresses a TAg that is unable to complex with p53 but retains N-terminal transforming functions, including the ability to complex pRB, p107, and p300. When SV40 TAgd11137 is expressed in villus enterocytes, they reenter into the cell cycle. However, coexpression of SV40 TAgd11137 and [Val12]KRAS does not produce dysplastic changes. Thus, the N-terminal 121 residues of TAg are sufficient to perturb the proliferative state of the enterocyte but not to produce detectable changes in the state of differentiation when coexpressed with [Val12]KRAS.
The nuclear localization signal of the matrix protein of human immunodeficiency virus type 1 allows the establishment of infection in macrophages and quiescent T lymphocytes.
Authors: von Schwedler U , Kornbluth R S , Trono D
Published: May 2006

Lentiviruses, including human immunodeficiency virus type 1 (HIV-1), are unusual among retroviruses in their ability to infect nondividing cells. The matrix proteins of several lentiviruses contain a short stretch of amino acids reminiscent of known nuclear localization signals. In HIV-1, this motif has been shown to function as a nuclear targeting sequence when conjugated to a heterologous protein, and to permit the active nuclear import of the HIV-1 preintegration complex in growth-arrested cells. In the present work, mutations were introduced in the matrix nuclear localization region of T-cell- and macrophage-tropic HIV-1 clones. The resulting viral mutants replicated with normal or even accelerated kinetics in dividing cells, including activated peripheral blood lymphocytes. However, in sharp contrast with wild-type virus, the mutants could not grow efficiently in terminally differentiated macrophages or establish a stable and inducible infection intermediate in unstimulated peripheral blood lymphocytes. Because macrophages represent a major viral reservoir in vivo, and because at any given time most T cells in the body are quiescent, these results strongly suggest that the karyophilic properties of the matrix protein are critical for the spread of the virus in HIV-infected individuals, and consequently for AIDS pathogenesis.
Atypical regions in large genomic DNA sequences.
Authors: Scherer S , McPeek M S , Speed T P
Published: May 2006

Large genomic DNA sequences contain regions with distinctive patterns of sequence organization. We describe a method using logarithms of probabilities based on seventh-order Markov chains to rapidly identify genomic sequences that do not resemble models of genome organization built from compilations of octanucleotide usage. Data bases have been constructed from Escherichia coli and Saccharomyces cerevisiae DNA sequences of > 1000 nt and human sequences of > 10,000 nt. Atypical genes and clusters of genes have been located in bacteriophage, yeast, and primate DNA sequences. We consider criteria for statistical significance of the results, offer possible explanations for the observed variation in genome organization, and give additional applications of these methods in DNA sequence analysis.
Mutations in ras genes in cells cultured from mouse skin tumors induced by ultraviolet irradiation.
Authors: Nishigori C , Wang S , Miyakoshi J , Sato M , Tsukada T , Yagi T , Imamura S , Takebe H
Published: May 2006

Mutations in ras oncogenes were detected in cultured cells of mouse skin tumors induced by near-UV irradiation. DNA extracted from the UV-induced tumor cells was transfected to golden hamster embryo cells, and focus-forming ability was confirmed in 22 of 26 cell strains, 15 of which had the repetitive mouse sequence. Mouse ras genes were detected in 10 of these 22 cell strains. Point mutations in the ras genes were at Ha-ras codon 13 (GGC-->GTC in two strains, GGC-->AGC in one strain), Ki-ras codon 61 (CAA-->GAA in two strains), and N-ras codon 61 (CAA-->CAT in two strains, CAA-->AAA in two strains). In one tumor cell strain no base change was directed. Most mutations occurred at dipyrimidine sites. Pyrimidine dimers or pyrimidine(6-4)pyrimidone photoproducts are the likely cause of the skin cancers. The base change occurred preferentially at G.C base pairs, and transversions predominated.
Structural analysis of chromosomal rearrangements associated with the developmental mutations Ph, W19H, and Rw on mouse chromosome 5.
Authors: Nagle D L , Martin-DeLeon P , Hough R B , Bućan M
Published: May 2006

We are studying the chromosomal structure of three developmental mutations, dominant spotting (W), patch (Ph), and rump white (Rw) on mouse chromosome 5. These mutations are clustered in a region containing three genes encoding tyrosine kinase receptors (Kit, Pdgfra, and Flk1). Using probes for these genes and for a closely linked locus, D5Mn125, we established a high-resolution physical map covering approximately 2.8 Mb. The entire chromosomal segment mapped in this study is deleted in the W19H mutation. The map indicates the position of the Ph deletion, which encompasses not more than 400 kb around and including the Pdgfra gene. The map also places the distal breakpoint of the Rw inversion to a limited chromosomal segment between Kit and Pdgfra. In light of the structure of the Ph-W-Rw region, we interpret the previously published complementation analyses as indicating that the pigmentation defect in Rw/+ heterozygotes could be due to the disruption of Kit and/or Pdgfra regulatory sequences, whereas the gene(s) responsible for the recessive lethality of Rw/Rw embryos is not closely linked to the Ph and W loci and maps proximally to the W19H deletion. The structural analysis of chromosomal rearrangements associated with W19H, Ph, and Rw combined with the high-resolution physical mapping points the way toward the definition of these mutations in molecular terms and isolation of homologous genes on human chromosome 4.
The chicken beta/epsilon-globin enhancer directs autonomously regulated, high-level expression of the chicken epsilon-globin gene in transgenic mice.
Authors: Foley K P , Pruzina S , Winick J D , Engel J D , Grosveld F , Fraser P
Published: May 2006

In transiently transfected chicken erythroid cells, beta-like globin gene switching is mediated through differential activation of the cis-linked embryonic epsilon- and adult beta-globin genes by a shared enhancer. Two underlying mechanisms have been proposed: (i) tissue- and stage-specific factors activate the beta-globin promoter in adult erythroid cells (autonomous regulation); and (ii) the epsilon-globin promoter, although transcriptionally competent in both embryonic and adult cells, is suppressed at the adult stage through competition with the beta-globin promoter for interaction with the enhancer (competitive regulation). Analyses of transgenic mice carrying the chicken beta/epsilon-globin locus demonstrated that both genes depended on the enhancer for erythroid expression, but only the epsilon-globin gene exhibited developmentally appropriate transcription at levels comparable to the endogenous mouse globin genes. Surprisingly, the chicken epsilon-globin gene also appeared to be autonomously regulated, as has been observed for human embryonic and fetal beta-like globin genes in transgenic mice. These results suggest that the chicken beta/epsilon-globin enhancer possesses either embryonic stage or epsilon-globin gene specificity when incorporated into the murine germ line.
Sequence divergence of the red and green visual pigments in great apes and humans.
Authors: Deeb S S , Jorgensen A L , Battisti L , Iwasaki L , Motulsky A G
Published: May 2006

We have determined the coding sequences of red and green visual pigment genes of the chimpanzee, gorilla, and orangutan. The deduced amino acid sequences of these pigments are highly homologous to the equivalent human pigments. None of the amino acid differences occurred at sites that were previously shown to influence pigment absorption characteristics. Therefore, we predict the spectra of red and green pigments of the apes to have wavelengths of maximum absorption that differ by < 2 nm from the equivalent human pigments and that color vision in these nonhuman primates will be very similar, if not identical, to that in humans. A total of 14 within-species polymorphisms (6 involving silent substitutions) were observed in the coding sequences of the red and green pigment genes of the great apes. Remarkably, the polymorphisms at 6 of these sites had been observed in human populations, suggesting that they predated the evolution of higher primates. Alleles at polymorphic sites were often shared between the red and green pigment genes. The average synonymous rate of divergence of red from green sequences was approximately 1/10th that estimated for other proteins of higher primates, indicating the involvement of gene conversion in generating these polymorphisms. The high degree of homology and juxtaposition of these two genes on the X chromosome has promoted unequal recombination and/or gene conversion that led to sequence homogenization. However, natural selection operated to maintain the degree of separation in peak absorbance between the red and green pigments that resulted in optimal chromatic discrimination. This represents a unique case of molecular coevolution between two homologous genes that functionally interact at the behavioral level.
Progressive growth in immunodeficient mice and host cell recruitment by mouse endothelial cells transformed by polyoma middle-sized T antigen: implications for the pathogenesis of opportunistic vascular tumors.
Authors: Garlanda C , Parravicini C , Sironi M , De Rossi M , Wainstok de Calmanovici R , Carozzi F , Bussolino F , Colotta F , Mantovani A , Vecchi A
Published: May 2006

A retroviral construct encoding polyoma middle-sized T antigen was used to generate transformed endothelial cell lines from heart (H5V), brain (B9V), and whole-embryo (E10V) of C57BL/6 mice. When injected into syngeneic recipients, H5V and the less studied B9V and E10V cells caused vascular tumors which, depending on the number of cells inoculated, regressed or progressed, leading to death of the host. When H5V cells were injected into immunodeficient mice, tumors were observed with inocula which did not form lesions in immunocompetent recipients and regression did not occur. Treatment with anti-LFA-1, anti-Thy-1.2, and anti-CD8 antibodies abolished rejection; anti-CD4 was a somewhat less effective inhibitor of resistance. Animals with progressive tumors exhibited secondary lesions in various organs with prominent skin involvement in nude mice. Histologically, the tumors had the appearance of a hemangioma, with areas resembling Kaposi sarcoma. Cells lining vascular lacunae had the morphological features of injected H5V cells. The lesions were characterized by prominent neovascularization and mononuclear cell infiltration. Southern blot hybridization analysis revealed that approximately 5% of the cells in the tumor mass were transplanted H5V cells. Thus, the H5V transformed endothelial line causes vascular lesions that are sustained to a large extent by recruitment of host cells and manifests full malignant behavior only in immunocompromised hosts. The hypothesis of a tumor sustained by a minute proportion of transformed cells, which recruit host elements and express full malignant behavior only in immunodeficient hosts, would account for several features of some vascular neoplasms in man.
Identification of immunosuppressant-induced apoptosis in a murine B-cell line and its prevention by bcl-x but not bcl-2.
Authors: Gottschalk A R , Boise L H , Thompson C B , Quintáns J
Published: May 2006

Cyclosporin A, FK-506, and rapamycin are immunosuppressants often used as pharmacological probes to study lymphocyte activation and physiological cell death (PCD). Because cyclosporin A and FK-506 are known to prevent PCD in T-cell hybridomas and thymocytes, we used these reagents, as well as rapamycin, to determine whether they alter the pathway leading to apoptosis in murine WEHI-231 cells following surface IgM cross-linking. We observed that the immunosuppressants themselves induced PCD in WEHI-231 cells, but only in sublines susceptible to anti-IgM-mediated apoptosis. PCD was preceded by growth arrest and characterized by the DNA fragmentation pattern typical of apoptosis. In B-cell lines resistant to anti-immunoglobulin- and immunosuppressant-induced PCD, cyclosporin A, FK-506, and rapamycin caused growth arrest. PCD was also induced by inhibitors of protein synthesis in WEHI-231 cells but not in the mature B-cell line BAL-17. Immunosuppressant-induced and protein synthesis inhibitor-induced PCD, but not growth arrest, could be prevented by the overexpression of bcl-xL, while transfection with bcl-2 did not affect PCD or cell cycle arrest. These results suggest that bcl-2 and bcl-xL may control partially independent systems to inhibit PCD in lymphoid cells and that PCD in B and T cells may be differentially regulated.
Climate change and temperature-dependent sex determination in reptiles.
Authors: Janzen F J
Published: May 2006

Despite increasing concern over the possible impact of global temperature change, there is little empirical evidence of direct temperature effects on biotic interactions in natural systems. Clear assessment of the ecological and evolutionary impact of changing climatic temperature requires a natural system in which populations exhibit a direct unambiguous fitness response to thermal fluctuation. I monitored nests of a population of painted turtles (Chrysemys picta) with temperature-dependent sex determination to investigate the causal relationship between local climatic variation in temperature and offspring sex ratio. Consistent with theoretical predictions, annual offspring sex ratio was highly correlated with mean July air temperature, validating concerns about the effect of climate change on population demography. This correlation implies that even modest increases in mean temperature (< 2 degrees C) may drastically skew the sex ratio. Statistical evaluation of the variance in climate change indicates that an increase in mean temperature of 4 degrees C would effectively eliminate production of male offspring. Quantitative genetic analyses and behavioral data suggest that populations with temperature-dependent sex determination may be unable to evolve rapidly enough to counteract the negative fitness consequences of rapid global temperature change. Populations of species with temperature-dependent sex determination may serve as ideal indicators of the biological impact of global temperature change.
HLA-D alleles associated with generalized disease, localized disease, and putative immunity in Onchocerca volvulus infection.
Authors: Meyer C G , Gallin M , Erttmann K D , Brattig N , Schnittger L , Gelhaus A , Tannich E , Begovich A B , Erlich H A , Horstmann R D
Published: May 2006

Human infections with the tissue nematode Onchocerca volvulus result in a variety of clinical conditions that possibly include protective immunity. In a West African area hyperendemic for human onchocerciasis, 120 residents were classified according to clinical and laboratory findings as presenting with generalized onchocerciasis, localized onchocerciasis, or as being putatively immune. The three groups differed in the distribution of HLA-D variants as determined by DNA typing. The most pronounced differences were found among alleles of the DQ loci. The haplotype DQA1*0501-DQB1*0301 was significantly more frequent among putatively immune individuals than among patients with generalized or localized disease. Conversely, DQA1*0101-DQB1*0501 and, independently, the allele DQB1*0201 were more frequent in generalized disease than in localized disease or putative immunity. In these correlations, the frequencies of allelic variants were in localized disease intermediate to those of the two other groups. The only distinct association found with localized disease was that of the DP allele DPB1*0402. The findings indicate that HLA-D variants influence the course of O. volvulus infection and help to define a state that may reflect protective immunity.
Localization of a putative tumor suppressor gene by using homozygous deletions in melanomas.
Authors: Weaver-Feldhaus J , Gruis N A , Neuhausen S , Le Paslier D , Stockert E , Skolnick M H , Kamb A
Published: May 2006

The p21 region of human chromosome 9 is thought to contain a gene (MLM) involved in genetic susceptibility to melanoma and a gene or genes that influence progression of certain other tumors. Genomic clones that span a large region in 9p21 surrounding the presumptive tumor suppressor gene(s) have been isolated. A set of sequence-tagged sites in this region has been developed. By using these markers and others previously reported, the 9p21 region has been studied by physical mapping in 84 melanoma cell lines. A putative tumor suppressor gene, perhaps MLM itself, has been localized to a region of less than 40 kb that lies proximal (centromeric) to the alpha-interferon gene cluster.