Important Publications at Cell

1 . Molecular Interactions between Bacterial Symbionts and Their HostsColin Dale and Nancy A. MoranCell 126: 453-465.[Full Text] [PDF]
Symbiotic bacteria are important in animal hosts, but have been largely overlooked as they have proved difficult to culture in the laboratory. Approaches such as comparative genomics and real-time PCR have provided insights into the molecular mechanisms that underpin symbiont-host interactions. Studies on the heritable symbionts of insects have yielded valuable information about how bacteria infect host cells, avoid immune responses, and manipulate host physiology. Furthermore, some symbionts use many of the same mechanisms as pathogens to infect hosts and evade immune responses. Here we discuss what is currently known about the interactions between bacterial symbionts and their hosts.
2 . Loss of ARNT/HIF1β Mediates Altered Gene Expression and Pancreatic-Islet Dysfunction in Human Type 2 DiabetesJenny E. Gunton, Rohit N. Kulkarni, SunHee Yim, Terumasa Okada, Wayne J. Hawthorne, Yu-Hua Tseng, Russell S. Roberson, Camillo Ricordi, Philip J. O’Connell, Frank J. Gonzalez and C. Ronald KahnCell 122: 337-349.[Full Text] [PDF]
β cell dysfunction is a central component of the pathogenesis of type 2 diabetes. Using oligonucleotide microarrays and real-time PCR of pancreatic islets isolated from humans with type 2 diabetes versus normal glucose-tolerant controls, we identified multiple changes in expression of genes known to be important in β cell function, including major decreases in expression of HNF4α, insulin receptor, IRS2, Akt2, and several glucose-metabolic-pathway genes. There was also a 90% decrease in expression of the transcription factor ARNT. Reducing ARNT levels in Min6 cells with small interfering RNA (siRNA) resulted in markedly impaired glucose-stimulated insulin release and changes in gene expression similar to those in human type 2 islets. Likewise, β cell-specific ARNT knockout mice exhibited abnormal glucose tolerance, impaired insulin secretion, and changes in islet gene expression that mimicked those in human diabetic islets. Together, these data suggest an important role for decreased ARNT and altered gene expression in the impaired islet function of human type 2 diabetes.
3 . From Microbes to Prions: The Final Proof of the Prion HypothesisWen-Quan Zou and Pierluigi GambettiCell 121: 155-157.[Full Text] [PDF]
Much like the “microbe hypothesis” put forth over 150 years ago, the “prion hypothesis” can be definitely proven only if a prion disease is engendered in a natural host from an infectious prion produced in vitro. In this issue of Cell, Akman et al., 2002Abbott et al., 2000Castilla et al., 2005Blackburn, 1992 come very close to accomplishing this goal by producing a prion disease in a natural host from a prion entirely generated in vitro using a PCR-like amplification system.
4 . Microarray Identification of FMRP-Associated Brain mRNAs and Altered mRNA Translational Profiles in Fragile X SyndromeVictoria Brown, Peng Jin, Stephanie Ceman, Jennifer C. Darnell, William T. O'Donnell, Scott A. Tenenbaum, Xiaokui Jin, Yue Feng, Keith D. Wilkinson, Jack D. Keene, Robert B. Darnell and Stephen T. WarrenCell 107: 477-487.[Full Text] [PDF]
Fragile X syndrome results from the absence of the RNA binding FMR protein. Here, mRNA was coimmunoprecipitated with the FMRP ribonucleoprotein complex and used to interrogate microarrays. We identified 432 associated mRNAs from mouse brain. Quantitative RT-PCR confirmed some to be >60-fold enriched in the immunoprecipitant. In parallel studies, mRNAs from polyribosomes of fragile X cells were used to probe microarrays. Despite equivalent cytoplasmic abundance, 251 mRNAs had an abnormal polyribosome profile in the absence of FMRP. Although this represents <2% of the total messages, 50% of the coimmunoprecipitated mRNAs with expressed human orthologs were found in this group. Nearly 70% of those transcripts found in both studies contain a G quartet structure, demonstrated as an in vitro FMRP target. We conclude that translational dysregulation of mRNAs normally associated with FMRP may be the proximal cause of fragile X syndrome, and we identify candidate genes relevant to this phenotype.
5 . RNAi as Random Degradative PCR: siRNA Primers Convert mRNA into dsRNAs that Are Degraded to Generate New siRNAsConcetta Lipardi, Qin Wei and Bruce M. PatersonCell 107: 297-307.[Full Text] [PDF]
In posttranscriptional gene silencing (PTGS), “quelling,” and RNA interference (RNAi), 21–25 nucleotide RNA fragments are produced from the initiating dsRNA. These short interfering RNAs (siRNAs) mediate RNAi by an unknown mechanism. Here, we show that GFP and Pp-Luc siRNAs, isolated from a protein complex in Drosophila embryo extract, target mRNA degradation in vitro. Most importantly, these siRNAs, as well as a synthetic 21-nucleotide duplex GFP siRNA, serve as primers to transform the target mRNA into dsRNA. The nascent dsRNA is degraded to eliminate the incorporated target mRNA while generating new siRNAs in a cycle of dsRNA synthesis and degradation. Evidence is presented that mRNA-dependent siRNA incorporation to form dsRNA is carried out by an RNA-dependent RNA polymerase activity (RdRP).
6 . Combinatorial Receptor Codes for OdorsBettina Malnic, Junzo Hirono, Takaaki Sato and Linda B Buck96: 713-723.[Full Text] [PDF]
The discriminatory capacity of the mammalian olfactory system is such that thousands of volatile chemicals are perceived as having distinct odors. Here we used a combination of calcium imaging and single-cell RT–PCR to identify odorant receptors (ORs) for odorants with related structures but varied odors. We found that one OR recognizes multiple odorants and that one odorant is recognized by multiple ORs, but that different odorants are recognized by different combinations of ORs. Thus, the olfactory system uses a combinatorial receptor coding scheme to encode odor identities. Our studies also indicate that slight alterations in an odorant, or a change in its concentration, can change its “code,” potentially explaining how such changes can alter perceived odor quality.
7 . Requirement for Specific Proteases in Cancer Cell Intravasation as Revealed by a Novel Semiquantitative PCR-Based AssayJ Kim, W Yu, K Kovalski and L Ossowski94: 353-362.[Full Text] [PDF]
Proteases are crucial for cancer metastasis, but due to lack of assays, their role in intravasation has not yet been tested. We have developed a human Alu sequence PCR-based assay to quantitate intravasated cells in an in vivo model. We demonstrated that metalloproteinases (MMPs), and most likely MMP-9, are required for intravasation by showing that marimastat, an inhibitor of MMPs, reduced intravasation by more than 90%, and that only tumor cell lines expressing MMP-9 intravasated. Cells with low surface urokinase plasminogen activator (uPA) and uPA receptor (uPAR) were also incapable of intravasation, despite the presence of high levels of MMP-9. We concluded that breaching of the vascular wall is a rate-limiting step for intravasation, and consequently for metastasis, and that cooperation between uPA/uPAR and MMP-9 is required to complete this step.
8 . Neandertal DNA Sequences and the Origin of Modern HumansMatthias Krings, Anne Stone, Ralf W Schmitz, Heike Krainitzki, Mark Stoneking and Svante Pääbo90: 19-30.[Full Text] [PDF]
DNA was extracted from the Neandertal-type specimen found in 1856 in western Germany. By sequencing clones from short overlapping PCR products, a hitherto unknown mitochondrial (mt) DNA sequence was determined. Multiple controls indicate that this sequence is endogenous to the fossil. Sequence comparisons with human mtDNA sequences, as well as phylogenetic analyses, show that the Neandertal sequence falls outside the variation of modern humans. Furthermore, the age of the common ancestor of the Neandertal and modern human mtDNAs is estimated to be four times greater than that of the common ancestor of human mtDNAs. This suggests that Neandertals went extinct without contributing mtDNA to modern humans.
9 . Altered Ca2+ Responses in Muscles with Combined Mitochondrial and Cytosolic Creatine Kinase DeficienciesKaren Steeghs, Ad Benders, Frank Oerlemans, Arnold de Haan, Arend Heerschap, Wim Ruitenbeek, Carolina Jost, Jan van Deursen, Benjamin Perryman, Dirk Pette, Marloes Brückwilder, Jolande Koudijs, Paul Jap, Jacques Veerkamp and Bé Wieringa89: 93-103.[Full Text] [PDF]
We have blocked creatine kinase (CK)-mediated phosphocreatine (PCr) ATP transphosphorylation in skeletal muscle by combining targeted mutations in the genes encoding mitochondrial and cytosolic CK in mice. Contrary to expectation, the PCr level was only marginally affected, but the compound was rendered metabolically inert. Mutant muscles in vivo showed significantly impaired tetanic force output, increased relaxation times, altered mitochondrial volume and location, and conspicuous tubular aggregates of sarcoplasmic reticulum membranes, as seen in myopathies with electrolyte disturbances. In depolarized myotubes cultured in vitro, CK absence influenced both the release and sequestration of Ca2+. Our data point to a direct link between the CK–PCr system and Ca2+-flux regulation during the excitation and relaxation phases of muscle contraction.
10 . Beyond PCR: Biotechnology as an Emerging CultureAdam Telerman and Robert B Amson86: 707-708.[Full Text] [PDF] 11 . The FHIT Gene at 3p14.2 Is Abnormal in Lung CancerGabriella Sozzi, Maria Luisa Veronese, Massimo Negrini, Raffaele Baffa, Maria Grazia Cotticelli, Hiroshi Inoue, Silvana Tornielli, Silvana Pilotti, Laura De Gregorio, Ugo Pastorino, Marco A Pierotti, Masataka Ohta, Kay Huebner and Carlo M Croce85: 17-26.[Full Text] [PDF]
To determine the role of the FHIT gene, which encompasses the fragile site at 3p14.2, we analyzed 59 tumors of the small cell and non-small cell type by reverse transcription of FHIT mRNA, followed by PCR amplification and sequencing of products. Allelic losses affecting the gene were evaluated by microsatellite polymorphism analysis and genomic alterations by hybridization using cDNA and genomic probes. Small cell lung tumors (80%) and non-small cell lung cancers (40%) showed abnormalities in RNA transcripts of FHIT, and 76% of the tumors exhibited loss of FHIT alleles. Abnormal lung tumor transcripts lack two or more exons of the FHIT gene. Small cell lung cancer tumors and cell lines were analyzed by Southern blotting and showed rearranged BamHI fragments. These data suggest a critical role of the FHIT gene in lung carcinogenesis.
12 . An in vitro assay for saccharomyces telomerase requires EST1Jing-Jer Lin and Virginia A. Zakian81: 1127-1135.[PDF]
Telomerase activity was demonstrated in cell-free extracts from S. cerevisiae through the use of a PCR-based assay. As expected, this activity was eliminated by RNase or phenol treatment of the extract and was dependent on dGTP and dTTP. Telomerase was not detected in extracts prepared from cells grown for ∼ 30 or more cell divisions in the absence of the EST1 product, Est1 p. TLC1 RNA, which determines the sequence of telomeric DNA in vivo, was present in normal amounts in est1Δ cells. Moreover, TLC1 RNA specifically precipitated with epitope-tagged Est1p. These data indicate that Estlp is either a subunit of yeast telomerase or an accessory protein associated with telomerase that is essential in vitro for its activity.
13 . A novel form of Epstein-Barr virus latency in normal B cells in vivoEmily M Miyashita, Bin Yang, Kitty M.C Lam, Dorothy H Crawford and David A Thorley-LawsonCell 80: 593-601.[PDF]
We have developed a PCR assay that can detect a single Epstein-Barr virus (EBV) genome in the presence of 106 uninfected cells. Using this assay, we demonstrate that EBV persists, in the peripheral blood of all seropositive individuals tested, in CD19+, CD23−, and CD80 (B7)− B cells. We further show that the virus in these cells is latent, but readily reactivated to produce infectious immortalizing virus; therefore, these cells represent a true site of latent persistence. EBV was not significantly detected in monocytes or T cells. The frequency of infected cells in nine healthy donors varied from 23 to 625 per 107 B cells, but was relatively stable for each individual over the course of 2 years. We conclude that the EBV-infected cells in vivo are B cells with a nonactivated phenotype. This represents a novel form of latency in normal B cells.
14 . The gene for neuronal apoptosis inhibitory protein is partially deleted in individuals with spinal muscular atrophyNatalie Roy, Mani S Mahadevan, Michael McLean, Gary Shutter, Zahra Yaraghi, Reza Farahani, Stephen Baird, Anne Besner-Johnston, Charles Lefebvre, Xiaolin Kang, Maysoon Salih, Huguette Aubry, Katsuyuki Tamai, Xiaoping Guan, Panayiotis Ioannou, Thomas O Crawford, Pieter J de Jong, Linda Surh, Joh-E Ikeda, Robert G Korneluk and Alex MacKenzieCell 80: 167-178.[PDF]
The spinal muscular atrophies (SMAs), characterized by spinal cord motor neuron depletion, are among the most common autosomal recessive disorders. One model of SMA pathogenesis invokes an inappropriate persistence of normally occurring motor neuron apoptosis. Consistent with this hypothesis, the novel gene for neuronal apoptosis inhibitory protein (NAIP) has been mapped to the SMA region of chromosome 5q13.1 and is homologous with baculoviral apoptosis inhibitor proteins. The two first coding exons of this gene are deleted in approximately 67% of type I SMA chromosomes compared with 2% of non-SMA chromosomes. Furthermore, RT-PCR. analysis reveals internally deleted and mutated forms of the NAIP transcript in type I SMA individuals and not in unaffected individuals. These findings suggest that mutations in the NAIP locus may lead to a failure of a normally occurring inhibition of motor neuron apoptosis resulting in or contributing to the SMA phenotype.
15 . HIV and T cell expansion in splenic white pulps is accompanied by infiltration of HIV-specific cytotoxic T lymphocytesRémi Cheynier, Sven Henrichwark, Fabienne Hadida, Eric Pelletier, Eric Oksenhendler, Brigitte Autran and Simon Wain-HobsonCell 78: 373-387.[PDF]
Human immunodeficiency virus (HIV) replication and T cell proliferation were investigated in situ by a PCR-based analysis of individual microdissected splenic white pulps. Founder effects, revealed by an exquisite compartmentalization of HIV genotypes and T cells, indicated the recruitment of latently infected CD4+ T cells through highly localized antigen presentation rather than the infection of CD4+ T lymphoblasts by blood-borne virus or immune complexes. HIV-infected white pulps could be infiltrated by HIV-specific cytotoxic T lymphocytes, thereby implicating them in CD4+ T cell destruction in vivo. Together these data describe an iterative and deleterious mechanism of antigen-driven T cell recruitment and activation, as well as HIV replication and spread, with consequent destruction of the newly infected cells.
16 . Skeletal muscles of mice deficient in muscle creatine kinase lack burst activityJan van Deursen, Arend Heerschap, Frank Oerlemans, Wim Rultenbeek, Paul Jap, Henk ter Laak and Bé WieringaCell 74: 621-631.[PDF]
To understand the physiological role of the creatine kinase-phosphocreatine (CK-PCr) system in muscle bioenergetics, a null mutation of the muscle CK (M-CK) gene was introduced into the germline of mice. Mutant mice show no alterations in absolute muscle force, but lack the ability to perform burst activity. Their fast-twitch fibers have an increased intermyofibrillar mitochondrial volume and an increased glycogenolytic/glycolytic potential. PCr and ATP levels are normal in resting M-CK-deficient muscles, but rates of high energy phosphate exchange between PCr and ATP are at least 20-fold reduced. Strikingly, PCr levels decline normally during muscle exercise, suggesting that M-CK-mediated conversion is not the only route for PCr utilization in active muscle.
17 . Hotspots for unselected Ty1 transposition events on yeast chromosome III are near tRNA genes and LTR sequencesH. Ji, D.P. Moore, M.A. Blomberg, L.T. Braiterman, D.F. Voytas, G. Natsoulis and J.D. BoekeCell 73: 1007-1018.[PDF]
A collection of yeast strains bearing single marked Ty1 insertions on chromosome III was generated. Over 100 such insertions were physically mapped by pulsed-field gel electrophoresis. These insertions are very nonrandomly distributed. Thirty-two such insertions were cloned by the inverted PCR technique, and the flanking DNA sequences were determined. The sequenced insertions all fell within a few very limited regions of chromosome III. Most of these regions contained tRNA coding regions and/or LTRs of preexisting transposable elements. Open reading frames were disrupted at a far lower frequency than expected for random transposition. The results suggest that the Ty1 integration machinery can detect regions of the genome that may represent “safe havens” for insertion. These regions of the genome do not contain any special DNA sequences, nor do they behave as particularly good targets for Ty1 integration in vitro, suggesting that the targeted regions have special properties allowing specific recognition in vivo.
18 . Mapping the whole human genome by fingerprinting yeast artificial chromosomesChristine Bellanné-Chantelot, Bruno Lacroix, Pierre Ougen, Alain Billault, Sandrine Beaufils, Stéphane Bertrand, Isabelle Georges, Fabrice Glibert, Isabelle Gros, Georges Lucotte, Laurent Susini, Jean-Jacques Codani, Philippe Gesnouin, Stuart Pook, Guy Vaysseix, Jennifer Lu-Kuo, Thomas Ried, David Ward, Ilya Chumakov, Denis Le Paslier, Emmanuel Barillot and Daniel CohenCell 70: 1059-1068.[PDF]
Physical mapping of the human genome has until now been envisioned through single chromosome strategies. We demonstrate that by using large insert yeast artificial chromosomes (YACs) a whole genome approach becomes feasible. YACs (22,000) of 810 kb mean size (5 genome equivalents) have been fingerprinted to obtain individual patterns of restriction fragments detected by a LINE-1 (L1) probe. More than 1000 contigs were assembled. Ten randomly chosen contigs were validated by metaphase chromosome fluorescence in situ hybridization, as well as by analyzing the inter-Alu PCR patterns of their constituent YACs. We estimate that 15% to 20% of the human genome, mainly the L1-rich regions, is already covered with contigs larger than 3 Mb.
19 . Molecular basis of myotonic dystrophy: Expansion of a trinucleotide (CTG) repeat at the 3′ end of a transcript encoding a protein kinase family memberJ.David Brook, Mila E. McCurrach, Helen G. Harley, Alan J. Buckler, Deanna Church, Hiroyuki Aburatani, Kent Hunter, Vincent P. Stanton, Jean-Paul Thirion, Thomas Hudson, Robert Sohn, Boris Zemelman, Russell G. Snell, Shelley A. Rundle, Steve Crow, June Davies, Peggy Shelbourne, Jessica Buxton, Clare Jones, Vesa Juvonen, Keith Johnson, Peter S. Harper, Duncan J. Shaw and David E. HousmanCell 68: 799-808.[PDF]
Using positional cloning strategies, we have identified a CTG triplet repeat that undergoes expansion in myotonic dystrophy patients. This sequence is highly variable in the normal population. PCR analysis of the interval containing this repeat indicates that unaffected individuals have between 5 and 27 copies. Myotonic dystrophy patients who are minimally affected have at least 50 repeats, while more severely affected patients have expansion of the repeat containing segment up to several kilobase pairs. The CTG repeat is transcribed and is located in the 3′ untranslated region of an mRNA that is expressed in tissues affected by myotonic dystrophy. This mRNA encodes a polypeptide that is a member of the protein kinase family.
20 . In vivo transfer of the human cystic fibrosis transmembrane conductance regulator gene to the airway epitheliumMelissa A. Rosenfeld, Kunihiko Yoshimura, Bruce C. Trapnell, Koichi Yoneyama, Eugene R. Rosenthal, Wilfried Dalemans, Masashi Fukayama, Joachim Bargon, Larue E. Stier, Leslie Stratford-Perricaudet, Michel Perricaudet, William B. Guggino, Andrea Pavirani, Jean-Pierre Lecocq and Ronald G. CrystalCell 68: 143-155.[PDF]
Direct transfer of the normal cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene to airway epithelium was evaluated using a replication-deficient recombinant adenovirus (Ad) vector containing normal human CFTR cDNA (Ad-CFTR). In vitro Ad-CFTR-infected CFPAC-1 CF epithelial cells expressed human CFTR mRNA and protein and demonstrated correction of defective cAMP-mediated Cl− permeability. Two days after in vivo intratracheal introduction of Ad-CFTR in cotton rats, in situ analysis demonstrated human CFTR gene expression in lung epithelium. PCR amplification of reverse transcribed lung RNA demonstrated human CFTR transcripts derived from Ad-CFTR, and Northern analysis of lung RNA revealed human CFTR transcripts for up to 6 weeks. Human CFTR protein was detected in epithelial cells using anti-human CFTR antibody 11–14 days after infection. While the safety and effectiveness remain to be demonstrated, these observations suggest the feasibility of in vivo CFTR gene transfer as therapy for the pulmonary manifestations of CF.
21 . Multipotent neural cell lines can engraft and participate in development of mouse cerebellumEvan Y. Snyder, David L. Deitcher, Christopher Walsh, Susan Arnold-Aldea, Erika A. Hartwieg and Constance L. CepkoCell 68: 33-51.[PDF]
Multipotent neural cell lines were generated via retrovirus-mediated v-myc transfer into murine cerebellar progenitor cells. When transplanted back into the cerebellum of newborn mice, these cells integrated into the cerebellum in a nontumorigenic, cytoarchitecturally appropriate manner. Cells from the same clonal line differentiated into neurons or glia in a manner appropriate to their site of engraftment. Engrafted cells, identified by lacZ expression and PCR-mediated detection of a unique sequence arrangement, could be identified in animals up to 22 months postengraftment. Electron microscopic and immunohistochemical analysis demonstrated that some engrafted cells were similar to host neurons and glia. Some transplant-derived neurons received appropriate synapses and formed normal intercellular contacts. These data indicate that generating immortalized cell lines for repair of, or transport of genes into, the CNS may be feasible. Such lines may also provide a model for commitment and differentiation of cerebellar progenitor cells.
22 . A gene encoding a protein serine/threonine kinase is required for normal development of M. xanthus, a gram-negative bacteriumJosé Muñoz-Dorado, Sumiko Inouye and Masayori InouyeCell 67: 995-1006.[PDF]
PCR reactions were carried out on the genomic DNA of M. xanthus, a soil bacterium capable of differentiation to form fruiting bodies, using oligonucleotides representing highly conserved regions of eukaryotic protein serine/threonine kinases. A gene (pkn1) thus cloned contains an ORF of 693 amino acid residues whose amino-terminal domain shows significant sequence similarity with the catalytic domain of eukaryotic protein serine/threonine kinases. The pkn1 gene was overexpressed in E. coli, and the gene product has been found to be autophosphorylated at both serine and threonine residues. The expression of pkn1 is developmentally regulated to start immediately before spore formation. When pkn1 is deleted, differentiation starts prematurely, resulting in poor spore production. These results indicate that the protein serine/threonine kinase plays an important role in the onset of proper differentiation.
23 . CREM gene: Use of alternative DNA-binding domains generates multiple antagonists of cAMP-induced transcriptionNicholas S. Foulkes, Emiliana Borrelli and Paolo Sassone-CorsiCell 64: 739-749.[PDF]
We isolated a gene from a mouse pituitary cDNA library that encodes a protein highly homologous to nuclear factor CREB, an activator of cAMP-responsive promoter elements (CREs). We demonstrate that while CREB is expressed uniformly in several cell types, this gene, termed CREM, shows cell-specific expression. CREM has a remarkable organization, since downstream of the stop codon there is a second, out-of-frame DNA-binding domain. Using PCR and RNAase protection analysis, we have identified three mRNA isoforms that appear to be obtained by differential cell-specific splicing. Sequencing of the isoforms demonstrated alternative usage of the two DNA-binding domains. CREM proteins reveal the same efficiency and specificity of binding to CRE sequences as CREB, but in contrast to CREB, CREM acts as a down-regulator of cAMP-induced transcription.
24 . Scrambled exonsJanice M. Nigro, Kathleen R. Cho, Eric R. Fearon, Scott E. Kern, J.Michael Ruppert, Jonathan D. Oliner, Kenneth W. Kinzler and Bert VogelsteinCell 64: 607-613.[PDF]
Using a sensitive assay for RNA expression, we identified several abnormally spliced transcripts in which exons from a candidate tumor suppressor gene (DCC) were scrambled during the splicing process in vivo. Cloning and sequencing of PCR-amplified segments of the abnormally spliced transcripts showed that exons were joined accurately at consensus splice sites, but in an order different from that present in the primary transcript. Four scrambled transcripts were identified, each involving a different pair of exons. The scrambled transcripts were found at relatively low levels in a variety of normal and neoplastic cells of rodent and human origin, primarily in the nonpolyadenylated component of cytoplasmic RNA. These results demonstrate that the splicing process does not always pair sequential exons in the order predicted from their positions in genomic DNA, thus creating a novel type of RNA product.
25 . Retrotransposition of a mouse IAP sequence tagged with an indicator geneOdile Heldmann and Thierry HeidmannCell 64: 159-170.[PDF]
We have marked a cloned mouse IAP sequence with a neomycin-containing indicator gene whose expression is conditioned by passage of the transposon through an RNA intermediate. Transposition of the marked IAP introduced into tumor cells could be detected by simple selection of the cells in G418, at a frequency of 10−6 per cell per generation. Southern blot analysis and nucleotide sequencing after PCR amplification demonstrated “retrotransposition” of the marked element, with spllcing out of an Intron contained in the indicator gene, and retroviral-like reverse transcription and integration of the transposed IAPs, with 6 bp dupiications of the identified target sites. Transposition was found to be mutagenic for the element, as might be expected if the identified marked and endogenous IAP transcripts were coencapsidated into IAP particies as dimers.
26 . Activin can induce the formation of axial structures and is expressed in the hypoblast of the chickE. Mitrani, T. Ziv, G. Thomsen, Y. Shimoni, D.A. Melton and A. BrilCell 63: 495-501.[PDF]
We show that PIF/activin can induce the formation of axial structures including a full-length notochord, segmented somites, and a neural tube in isolated epiblasts from chick blastulae. Using degenerate PCR primers, we have cloned a fragment of the activin βB chain from chick hypobiast cDNA, and a fragment of the activin βA chain from chick genomic DNA. Furthermore, we show that in the chick, activin is transcribed precisely when axial mesoderm is being induced. Since exogenous PIF/activin can induce the formation of axial structures and since activin βB is transcribed at the time and place where the mesodermal axial structures are being induced, we propose that in the chick, activin B is the endogenous inducer of the body axis.
27 . A major segment of the neurofibromatosis type 1 gene: cDNA sequence, genomic structure, and point mutationsRichard M. Cawthon, Robert Weiss, Gangfeng Xu, David Viskochil, Melanie Culver, Jeff Stevens, Margaret Robertson, Diane Dunn, Ray Gesteland, Peter O'Connell and Ray WhiteCell 62: 193-201.[PDF]
Overlapping cDNA clones from the translocation break-point region (TBR) gene, recently discovered at the neurofibromatosis type 1 locus and found to be interrupted by deletions and a t(17;22) translocation, have been sequenced. A 4 kb sequence of the transcript of the TBR gene has been compared with sequences of genomic DNA, identifying a number of small exons. Identification of splice junctions and a large open reading frame indicates that the gene is oriented with its 5′ end toward the centromere, in opposition to the three known active genes in the region. PCR amplification of a subset of the exons, followed by electrophoresis of denatured product on native gels, identified six variant conformers specific to NF1 patients, indicating base pair changes in the gene. Sequencing revealed that one mutant allele contains a T→C transition changing a leucine to a proline; another NF1 allele harbors a C→T transition changing an arginine to a stop codon. These results establish the TBR gene as the NF1 gene and provide a description of a major segment of the gene.
28 . Chromosomal translocation t(1;19) results in synthesis of a homeobox fusion mRNA that codes for a potential chimeric transcription factorJamison Nourse, Julia D. Mellentin, Naomi Galili, Joyce Wilkinson, Eric Stanbridge, Stephen D. Smith and Michael L. ClearyCell 60: 535-545.[PDF]
The gene (E2A) for enhancer binding transcription factors E12 and E47 maps to the t(1;19) chromosomal translocation breakpoint in pre-B cell leukemias. Altered E2A transcripts lacking sequences coding for the helix-loop-helix DNA binding motif were detected in several t(1;19)-carrying cell lines. Fusion cDNAs that crossed the t(1;19) breakpoint were cloned and shown to code for an 85 kd protein consisting of the amino-terminal two-thirds of E2A fused to a chromosome 1-derived protein. The fusion protein has the features of a chimeric transcription factor in which the DNA binding domain of E2A is replaced by the putative DNA binding domain of a homeoprotein from chromosome 1 for which the name Prl (pre-B cell leukemia) is proposed. Identical E2A-prl mRNA junctions were detected by PCR in three t(1;19)-carrying cell lines, indicating that the fusion transcripts and predicted chimeric protein are a consistent feature of this translocation.
29 . Temporal fluctuations in HIV quasispecies in vivo are not reflected by sequential HIV isolationsAndreas Meyerhans, Rémi Cheynier, Jan Albert, Martina Seth, Shirley Kwok, John Sninsky, Linda Morfeldt-Månson, Birgitta Asjö and Simon Wain-HobsonCell 58: 901-910.[PDF]
A genetic study has been made of the HIV tat gene from sequential HIV-1 isolates and the corresponding infected peripheral blood mononuclear cells. DNA was amplified by polymerase chain reaction (PCR) and cloned into a eukaryotic expression vector. Twenty clones were sequenced from each sample. Comparing the sequential HIV isolates, abrupt differences were seen between the major forms of each isolate. These progressive changes were not reflected at all among the in vitro samples. The fluctuation in the quasispecies in vivo may suggest a much more dynamic role for Iatently infected mononuclear cells. High frequencies of functionally defective tat genes were identified. Given such complexity and the evident differences between quasispecies in vivo and in vitro, the task of defining HIV infection in molecular terms will be difficult.
1 . A Short Primer on RNAi: RNA-Directed RNA Polymerase Acts as a Key Catalyst
Kazuko NishikuraCell 107: 415-418.[Full Text] [PDF]
One of the many intriguing features of gene silencing by RNA interference is the apparent catalytic nature of the phenomenon. New biochemical and genetic evidence now shows that an RNA-directed RNA polymerase chain reaction, primed by siRNA, amplifies the interference caused by a small amount of “trigger” dsRNA.
2 . Nucleosomes, DNA-binding proteins, and DNA sequence modulate retroviral integration target site selection
Peter M. Pryciak and Harold E. VarmusCell 69: 769-780.[PDF]
Integration of retroviral DNA can serve as a paradigm for cellular functions that are affected by the packaging of DNA into chromatin. We have used a novel polymerase chain reaction-based assay to survey DNA and chromatin for the precise distribution of many integration sites. Integration into naked DNA targets is nonuniform, implying a nucleotide sequence bias. In chromatin, integration occurs preferentially at postitions where the major groove is on the exposed face of the nucleosomal DNA helix, generating a 10 bp periodic spacing of preferred sites. Chromatin assembly enhances the reactivity of many sites, so that integration occurs most frequently at sites in nucleosomal, rather than nucleosome-free, regions of minichromosomes. In contrast, integration is prevented in a region occupied by a site-specific DNA-binding protein. Comparisons of integration events mediated by viral nucleoprotein complexes or by two different retroviral integrases show that the integration machinery also affects target site selection.
3 . Ubiquitous MyoD transcription at the midblastula transition precedes induction-dependent MyoD expression in presumptive mesoderm of X. laevis
Ralph A.W. Rupp and Harold WeintraubCell 65: 927-937.[PDF]
We have used a quantitative reverse transcription-polymerase chain reaction assay to detect MyoD mRNA during early embryonic development of Xenopus laevis. We find that during a short period of time following the midblastula transition MyoD becomes transcriptionally activated at a low level ubiquitously throughout the embryo. Restriction of MyoD expression to muscle precursor cells appears as a subsequent event, in which the process of mesoderm induction stabilizes transcription only in the marginal zone of the embryo, the presumptive mesoderm.
4 . Chimeric gRNA-mRNA molecules with oligo(U) tails covalently linked at sites of RNA editing suggest that U addition occurs by transesterification
Beat Blum, Nancy R. Sturm, Agda M. Simpson and Larry SimpsonCell 65: 543-550.[PDF]
Chimeric RNA molecules were detected by polymerase chain reaction ampllfication of kinetoplast RNA using a 3′ primer specific to mRNA and a 5′ primer specific to guide RNA (gRNA), and directly by Northern analysis. Covalent linkage of the 3′ oligo(U) tail of the gRNA to the mRNA occurs at editing sites. Chimeric molecules were isolated for NADH dehydrogenase subunit 7 and cytochrome oxidase subunits II and III. We propose that these molecules are intermediates in the editing process and that successive transesterifications resuit in the transfer of uridine residues from the gRNA 3′ oligo(U) tail to an editing site, with the number of uridine residues determined by base pairing with adenine and guanine “guide” nucleotides in the gRNA.
5 . A cyclin B homolog in S. cerevisiae: Chronic activation of the Cdc28 protein kinase by cyclin prevents exit from mitosis
Jayant B. Ghiara, Helena E. Richardson, Katsunori Sugimoto, Martha Henze, Daniel J. Lew, Curt Wittenberg and Steven I. ReedCell 65: 163-174.[PDF]
A cyclin B homolog was identified in Saccharomyces cerevisiae using degenerate oligonucleotides and the polymerase chain reaction. The protein, designated Scb1, has a high degree of similarity with B-type cyclins from organisms ranging from fission yeast to human. Levels of SCB1 mRNA and protein were found to be periodic through the cell cycle, with maximum accumulation late, most likely in the G2 interval. Deletion of the gene was found not to be lethal, and subsequently other B-type cyclins have been found in yeast functionally redundant with Scb1. A mutant allele of SCB1 that removes an amino-terminal fragment of the encoded protein thoughtto be required for efficient degradation during mitosis confers a mitotic arrest phenotype. This arrest can be reversed by inactivation of the Cdc28 protein kinase, suggesting that cyclin-mediated arrest results from persistent protein kinase activation.
6 . The recombination activating gene-1 (RAG-1) transcript is present in the murine central nervous system
Jerold J.M. Chun, David G. Schatz, Marjorie A. Oettinger, Rudolf Jaenisch and David BaltimoreCell 64: 189-200.[PDF]
The recombination activating genes, RAG-1 and RAG-2, are likely to encode components of the V(D)J site-specific recombination machinery. We report here the detection of low levels of the RAG-1 transcrlpt in the murlne central nervous system by polymerase chain reaction, In situ hybridization, and Northern blot analyses. In contrast, an authentic RAG-2 transcript could not be detected reproduclbly in the central nervous system. The RAG-1 transcript was found to be wide-spread in embryonic and postnatal neurons, with transcription being most apparent in regions of the postnatal brain with a high neuronal cell denslty (the cerebellum and the hippocampal formation). The results suggest that RAG-1 functions in neurons, where its role might be to recombine elements of the neuronal genome site-specifically, or to prevent detrimental alterations of the genome in these long-lived cells.
7 . Partially edited mRNAs for cytochrome b and subunit III of cytochrome oxidase from leishmania tarentolae mitochondria: RNA editing intermediates
Nancy R. Sturm and Larry SimpsonCell 61: 871-878.[PDF]
Partially edited mRNAs were selected by the polymerase chain reaction and sequenced. In the case of cytochrome b, 102 out of 106 clones displayed patterns of editing that were consistent with a strictly progressive 3′ to 5′ editing process, as predicted by the guide RNA model of RNA editing. In the case of cytochrome oxidase subunit III (COIII), 177 out of 304 clones displayed strictly progressive 3′ to 5′ patterns of editing. However, the remaining 127 COIII clones displayed unexpected patterns in which upstream editing preceded downstream editing, uridines were inserted at sites not normally edited, and purine residues were deleted. We suggest that many of these RNAs are produced by normal 3′ to 5′ editing of the COIII mRNA with incorrect guide RNA molecules.
8 . Molecular cloning and expression of the human 55 kd tumor necrosis factor receptor
Hansruedi Loetscher, Yu-Ching E. Pan, Hans-Werner Lahm, Reiner Gentz, Manfred Brockhaus, Hisahiro Tabuchi and Werner LesslauerCell 61: 351-359.[PDF]
Two distinct receptors for tumor necrosis factor (TNF) of 55 and 75 kd are expressed at low levels by various cells. The 55 kd TNF receptor was purified from HL60 cells, and partial amino acid sequences were determined. Short degenerate sense and antisense oligonucleotide primers encoding the N- and C-terminal ends of a peptide of 22 amino acid residues were used to amplify a 66 bp cDNA fragment from HL60 RNA by reverse transcriptase-polymerase chain reaction. The cDNA fragment as a probe identified several overlapping clones in a human placenta cDNA library. The open reading frame of the cDNA predicts a 455 amino acid TNF receptor protein with leader, extracellular, transmembrane, and intracellular domains. When expressed in COS-1 cells or in a baculovirus system, the cDNA conferred TNF binding properties comparable to the native receptor. Surprisingly, the 55 kd TNF receptor shows a high degree of sequence homology to the NGF receptor extracellular domain.
9 . HIV-1 entry into quiescent primary lymphocytes: Molecular analysis reveals a labile, latent viral structure
Jerome A. Zack, Salvatore J. Arrigo, Stacy R. Weitsman, Alan S. Go, Allyson Haislip and Irvin S.Y. ChenCell 61: 213-222.[PDF]
Productive infection of human T lymphocytes by HIV-1 is dependent upon proliferation of the infected cell. Nonproliferating quiescent T cells can be infected by HIV-1 and harbor the virus in an inactive state until subsequent mitogenic stimulation. We use a modification of the polymerase chain reaction method, which is both sensitive and quantitative, to demonstrate that HIV-1 DNA synthesis is initiated in infected quiescent T cells at levels comparable with those of activated T cells. However, unlike that of activated T cells, the viral genome is not completely reverse transcribed in quiescent cells. Although this viral DNA structure can persist in quiescent cells as a latent form, it is labile. We discuss the lability of this HIV-1 DNA structure in relation to a “self-restricting persistent infection” by HIV-1 and propose that this may explain the low percentage of infected cells in the circulation of AIDS patients.
10 . Detection of circular forms of eliminated DNA during macronuclear development in E. crassus
S.Lorraine Tausta and Lawrence A. KlobutcherCell 59: 1019-1026.[PDF]
Following their sexual cycle, hypotrichous ciliated protozoa transform a copy of a chromosomal micronucleus into a macronucleus containing small, linear DNA molecules. A frequent event during macronuclear development is the removal of short segments of DNA (internal eliminated sequences: IESs) by a process equivalent to DNA breakage and rejoining. In this study we used a polymerase chain reaction procedure to demonstrate that free circular forms of IESs are present in cells undergoing macronuclear development. Sequencing of the junctions of the free circular IESs suggests that they share 12 nucleotides with the macronuclear DNA molecules that are generated following IES removal. The results provide evidence that IESs are removed by an active DNA breakage and rejoining process, which may involve staggered cuts in the substrate DNA.
11 . Activation of immunoglobulin kappa gene rearrangement correlates with induction of germline kappa gene transcription
Mark S. Schlissel and David BaltimoreCell 58: 1001-1007.[PDF]
We have developed a sensitive polymerase chain reaction assay for measuring the fraction of rearranged immunoglobulin kappa genes in a cell population. Using this assay with Abelson virus-transformed murine pre-B cells, we have found that bacterial lipopolysacharide treatment, which activates transcription of the unrearranged kappa constant region gene, also activates kappa gene rearrangement. In addition, we have been able to detect kappa gene rearrangement in cell lines that do not produce a functional heavy chain gene product (mu protein). These results implicate transcription or transcription factor binding as a regulator of immunoglobulin gene rearrangement.
12 . Temporal fluctuations in HIV quasispecies in vivo are not reflected by sequential HIV isolations
Andreas Meyerhans, Rémi Cheynier, Jan Albert, Martina Seth, Shirley Kwok, John Sninsky, Linda Morfeldt-Månson, Birgitta Asjö and Simon Wain-HobsonCell 58: 901-910.[PDF]
A genetic study has been made of the HIV tat gene from sequential HIV-1 isolates and the corresponding infected peripheral blood mononuclear cells. DNA was amplified by polymerase chain reaction (PCR) and cloned into a eukaryotic expression vector. Twenty clones were sequenced from each sample. Comparing the sequential HIV isolates, abrupt differences were seen between the major forms of each isolate. These progressive changes were not reflected at all among the in vitro samples. The fluctuation in the quasispecies in vivo may suggest a much more dynamic role for Iatently infected mononuclear cells. High frequencies of functionally defective tat genes were identified. Given such complexity and the evident differences between quasispecies in vivo and in vitro, the task of defining HIV infection in molecular terms will be difficult.
13 . Catalytic deficiency of human aldolase B in hereditary fructose intolerance caused by a common missense mutation
Nicholas C.P. Cross, Dean R. Tolan and Timothy M. CoxCell 53: 881-885.[PDF]
Hereditary fructose intolerance (HFI) is a human autosomal recessive disease caused by a deficiency of aldolase B that results in an inability to metabolize fructose and related sugars. We report here the first identification of a molecular lesion in the aldolase B gene of an affected individual whose defective protein has previously been characterized. The mutation is a G→C transversion in exon 5 that creates a new recognition site for the restriction enzyme Ahall and results in an amino acid substitution (Ala→Pro) at position 149 of the protein within a region critical for substrate binding. Utilizing this novel restriction site and the polymerase chain reaction, the patient was shown to be homozygous for the mutation. Three other HFI patients from pedigrees unrelated to this individual were found to have the same mutation: two were homozygous and one was heterozygous. We suggest that this genetic lesion is a prevailing cause of hereditary fructose intolerance.
14 . Most human carcinomas of the exocrine pancreas contain mutant c-K-ras genes
Concepcion Almoguera, Darryl Shibata, Kathleen Forrester, John Martin, Norman Arnheim and Manuel PeruchoCell 53: 549-554.[PDF]
Using in vitro gene amplification by the polymerase chain reaction (PCR) and mutation detection by the RNAase A mismatch cleavage method, we have examined, c-K-ras genes in human pancreatic carcinomas. We used frozen tumor specimens and single 5 μm sections from formalin-fixed, paraffin-embedded tumor tissue surgically removed or obtained at autopsy. Twenty-one out of 22 carcinmas of the exocrine pancreas contained c-K-ras genes with mutations at codon 12. In seven cases tested, the mutation was present in both primary tumors and their corresponding metastases. from the same cancer patients or in five gall bladder carcinomas. We conclude from these results that c-K-ras somatic mutational activation is a critical event in the oncogenesis of most, if not all, human cancers of the exocrine pancreas.