bluuebunny/crossref_metadata_2025 huggingface dataset:
The TUBEX test detects not only typhoid-specific antibodies but also soluble antigens and whole bacteria
Authors: Tam Frankie C. H. , Ling Thomas K. W. , Wong Kam Tak , Leung Danny T. M. , Chan Raphael C. Y. , Lim Pak Leong
Published: February 2008

TUBEX (IDL Biotech) is a 5 min semiquantitative colorimetric test for typhoid fever, a widely endemic disease. TUBEX detects anti-Salmonella O9 antibodies from a patient's serum by the ability of these antibodies to inhibit the binding between an indicator antibody-bound particle and a magnetic antigen-bound particle. Herein, we report that TUBEX could also be used to specifically detect soluble O9 lipopolysaccharide in antigen-spiked buffer by the ability of the antigen to inhibit the same binding between the particles. Sensitivity of antigen detection was improved (8–31 μg ml−1) by using a modified protocol in which the test sample was mixed with the indicator particles first, rather than with the magnetic particles as for antibody detection. The antigen was also detectable in spiked serum and urine samples, albeit less well (2–4-fold) than in buffer generally. However, no antigen was detected from six typhoid sera examined, all of which had anti-O9 antibodies. In addition, whole organisms of Salmonella Typhi (15 strains) and Salmonella Enteritidis (6 strains) (both O9+
Salmonella), grown in simulated blood broths or on MacConkey agar, were also detectable by TUBEX when suspended at >9×108 organisms ml−1. Expectedly, Salmonella Paratyphi A (7 strains), Salmonella Typhimurium (1 strain) and Escherichia coli (2 strains) were negative in the test. Thus, the same TUBEX kit may be used in several ways both serologically and microbiologically for the rapid diagnosis of typhoid fever. However, validation of the newer applications will require the systematic examination of real patient and laboratory materials.
Isolates of Clostridium perfringens recovered from Costa Rican patients with antibiotic-associated diarrhoea are mostly enterotoxin-negative and susceptible to first-choice antimicrobials
Authors: Camacho Natassia , Espinoza Carlos , Rodríguez César , Rodríguez Evelyn
Published: February 2008

To assess the prevalence of enterotoxigenic Clostridium perfringens among adults suffering from antibiotic-associated diarrhoea in a Costa Rican hospital, faecal samples were analysed from 104 patients by a cultivation approach. The 29 strains obtained, which accounted for an isolation frequency of 28 %, were genotyped and investigated with regard to their in vitro susceptibility to penicillin, imipenem, cefotaxime, chloramphenicol and metronidazole using an agar-dilution method. A multiplex PCR for detection of the toxins α, β and ϵ predictably classified all faecal isolates as biotype A. An agglutination assay revealed that only one isolate synthesized detectable amounts of enterotoxin (detection rate 3 %). This result was confirmed by a PCR targeting the cpe gene. The spores of the only CPE+ isolate did not germinate after incubation for 30 min at temperatures above 80 °C. Most isolates were susceptible to first-choice antimicrobials. However, unusual MICs for penicillin (16 μg ml−1) and metronidazole (512 μg ml−1) were detected in one and three isolates, respectively. The low incidence of enterotoxigenic strains suggests that C. perfringens was not a major primary cause of antibiotic-associated diarrhoea in this hospital during the sampling period.
Glycosyltransferase – a specific marker for the discrimination of Bacillus anthracis from the Bacillus cereus group
Authors: Kim Wonyong , Kim Ji-Yeon , Cho Sung-Lim , Nam Sun-Woo , Shin Jong-Wook , Kim Yang-Soo , Shin Hyoung-Shik
Published: February 2008

Bacillus anthracis, the aetiological agent of anthrax, has been taxonomically classified with the Bacillus cereus group, which comprises B. cereus, Bacillus thuringiensis, Bacillus mycoides, Bacillus pseudomycoides and Bacillus weihenstephanensis. Although the pathogenesis and ecological manifestations may be different, B. anthracis shares a high degree of DNA sequence similarity with its group member species. As a result, the discrimination of B. anthracis from its close relatives in the B. cereus group is still quite difficult. Suppression subtractive hybridization (SSH) was performed to search for genomic differences between a B. anthracis Korean isolate CR and the most closely related B. cereus type strain KCTC 3624T. Two-hundred and five B. anthracis CR clones obtained by SSH underwent Southern hybridization, and comparative sequences were analysed using the blast program from the National Center for Biotechnology Information (NCBI). Subsequently, primer sets based on the glycosyltransferase group 1 family protein gene specific to B. anthracis were designed from the sequences of subtracted clones, and their specificities were evaluated using eight B. anthracis, 33 B. cereus, 10 B. thuringiensis, six B. mycoides, one B. pseudomycoides, one B. weihenstephanensis and 19 strains from 11 other representative Bacillus species. PCR primers specific for the glycosyltransferase group 1 family protein gene did not amplify the desired products from any of the Bacillus strains under examination, except B. anthracis alone. These findings may be useful in the future development of efficient diagnostic tools for the rapid identification of B. anthracis from other members of the B. cereus group.
PPARγ induces cell cycle withdrawal: inhibition of E2F/DP DNA-binding activity via down-regulation of PP2A
Authors: Altiok Soner , Xu Min , Spiegelman Bruce M.
Published: February 2008

PPARγ is an adipose-selective nuclear hormone receptor that plays a key role in the control of adipocyte differentiation. Previous studies indicated that activation of ectopically expressed PPARγ induces differentiation when cells have ceased growth because of confluence. We show here that ligand activation of PPARγ is sufficient to induce growth arrest in fibroblasts and SV40 large T-antigen transformed, adipogenic HIB1B cells. Cell cycle withdrawal is accompanied by a decrease in the DNA-binding and transcriptional activity of the E2F/DP complex, which is attributable to an increase in the phosphorylation of these proteins, especially DP-1. This effect is a consequence of decreased expression of the catalytic subunit of the serine–threonine phosphatase PP2A. These data suggest an important role for PP2A in the control of E2F/DP activity and a new mode of cell cycle control in differentiation.
Overexpression of Stra13, a novel retinoic acid-inducible gene of the basic helix–loop–helix family, inhibits mesodermal and promotes neuronal differentiation of P19 cells
Authors: Boudjelal Mohamed , Taneja Reshma , Matsubara Shyuichiro , Bouillet Philippe , Dollé Pascal , Chambon Pierre
Published: February 2008

We report the cDNA cloning of Stra13, a novel retinoic acid (RA)-inducible gene from P19 embryonal carcinoma cells that encodes a basic helix–loop–helix (bHLH) protein that shows the highest sequence similarities to theDrosophilaHairy and Enhancer of split and mouse Hes proteins. Stra13 does not bind to the known consensus motifs (E-box and N-box) for bHLH proteins, but can repress activated transcription (through an α-helix rich domain) in part by interaction with general factors of the basal transcription machinery. During mouse embryogenesis, Stra13 RNA is expressed in the neuroectoderm, and also in a number of mesodermal and endodermal derivatives. Remarkably, overexpression of Stra13 in P19 cells results in neuronal differentiation in monolayer culture, under conditions where wild-type P19 cells only undergo mesodermal/endodermal differentiation. This neuronal differentiation is accompanied by an altered expression of mesodermal and neuronal markers, indicating that Stra13 could be one of the earliest RA target genes whose expression is required for repression of mesodermal/endodermal differentiation and/or induction of neuronal differentiation when P19 cell aggregates are exposed to RA. Our results raise the possibility that Stra13 could be involved as a repressor in a number of decision events occurring during differentiation of various cell lineages.
Inhibition of CDK activity and PCNA-dependent DNA replication by p21 is blocked by interaction with the HPV-16 E7 oncoprotein
Authors: Funk Jens Oliver , Waga Shou , Harry Jo Beth , Espling Erik , Stillman Bruce , Galloway Denise A.
Published: February 2008

p21 inhibits cyclin-dependent kinase (CDK) activity and proliferating cell nuclear antigen (PCNA)-dependent DNA replication by binding to CDK/cyclin complexes and to PCNA through distinct domains. The human papillomavirus (HPV)-16 E7 oncoprotein (16E7) abrogated a DNA damage-induced cell cycle arrest in vivo, despite high levels of p21. Using cell lysates and purified proteins we show that 16E7 prevented p21 both from inhibiting CDK2/cyclin E activity and PCNA-dependent DNA replication, whereas the nononcogenic HPV-6 E7 had reduced effects. Inactivation of both inhibitory functions of p21 was attained through binding between 16E7 and sequences in the carboxy-terminal end of p21 that overlap with the PCNA-binding site and the second p21 cyclin-binding motif. These data imply that the carboxyl terminus of p21 simultaneously modulates both CDK activity and PCNA-dependent DNA replication and that a single protein, 16E7, can override this modulation to disrupt normal cell cycle control.
The human papillomavirus E7 oncoprotein can uncouple cellular differentiation and proliferation in human keratinocytes by abrogating p21Cip1-mediated inhibition of cdk2
Authors: Jones D. Leanne , Alani Rhoda M. , Münger Karl
Published: February 2008

The high risk human papillomaviruses (HPVs) are associated etiologically with the majority of human cervical carcinomas. These HPVs encode two viral oncoproteins, E6 and E7, which are expressed consistently in cervical cancers. The function of these viral oncoproteins during a productive infection is to ensure viral replication in cells that have normally withdrawn from the cell division cycle and are committed to terminal differentiation. Expression of the E7 oncoprotein has been shown to lead to the abrogation of various negative growth regulatory signals, including a p53-mediated G1 growth arrest, TGFβ-mediated growth inhibition, and quiescence of suprabasal keratinocytes. Here we describe a novel mechanism by which E7 can uncouple cellular proliferation and differentiation. In contrast to normal, differentiating keratinocytes, HPV-16 E7-expressing keratinocytes show delayed cellular differentiation and elevated cdk2 kinase activity despite high levels of p21Cip1 and association of p21Cip1 with cdk2. We show that the HPV E7 protein can interact with p21Cip1 and abrogate p21Cip1-mediated inhibition of cyclin A and E-associated kinase activities. Based on these findings, we propose that this capacity of the HPV E7 oncoprotein to overcome p21Cip1-mediated inhibition of cdk2 activity during keratinocyte differentiation contributes to the ability of E7 to allow for cellular DNA synthesis in differentiated keratinocytes.
Epigenetic silencing of RNA polymerase I transcription: a role for DNA methylation and histone modification in nucleolar dominance
Authors: Chen Z. Jeffrey , Pikaard Craig S.
Published: February 2008

Nucleolar dominance is an epigenetic phenomenon that describes nucleolus formation around rRNA genes inherited from only one progenitor of an interspecific hybrid or allopolyploid. The phenomenon is widespread, occurring in plants, insects, amphibians, and mammals, yet its molecular basis remains unclear. We have demonstrated nucleolar dominance in three allotetraploids of the plant genusBrassica.InBrassica napus,accurately initiated pre-rRNA transcripts from one progenitor,Brassica rapaare detected readily, whereas transcripts from the ∼3000 rRNA genes inherited from the other progenitor,Brassica oleracea,are undetectable. Nuclear run-on confirmed that dominance is controlled at the level of transcription. Growth ofB. napusseedlings on 5-aza-2′-deoxycytidine to inhibit cytosine methylation caused the normally silent, under-dominantB. oleracearRNA genes to become expressed to high levels. The histone deacetylase inhibitors sodium butyrate and trichostatin A also de-epressed silent rRNA genes. These results reveal an enforcement mechanism for nucleolar dominance in which DNA methylation and histone modifications combine to regulate rRNA gene loci spanning tens of megabase pairs of DNA.
Diabetes, defective pancreatic morphogenesis, and abnormal enteroendocrine differentiation in BETA2/NeuroD-deficient mice
Authors: Naya Francisco J. , Huang Hsiang-Po , Qiu Yuhong , Mutoh Hiroyuki , DeMayo Francesco J. , Leiter Andrew B. , Tsai Ming-Jer
Published: February 2008

Candidate transcription factors involved in pancreatic endocrine development have been isolated using insulin gene regulation as a paradigm. The cell-type restricted basic helix–loop–helix (bHLH) gene, BETA2/NeuroD, expressed in pancreatic endocrine cells, the intestine, and the brain, activates insulin gene transcription and can induce neurons to differentiate. To understand the importance of BETA2 in pancreatic endocrine cell differentiation, mice lacking a functional BETA2 gene were generated by gene targeting experiments. Mice carrying a targeted disruption of theBETA2 gene developed severe diabetes and died perinatally. Homozygous BETA2 null mice had a striking reduction in the number of insulin-producing β cells and failed to develop mature islets. Islet morphogenesis appeared to be arrested between E14.5 and E17.5, a period characterized by major expansion of the β cell population. The presence of severe diabetes in these mice suggests that proper islet structure plays an important role in blood glucose homeostasis. In addition, secretin- and cholecystokinin-producing enteroendocrine cells failed to develop in the absence of BETA2. The absence of these two pancreatic secretagogs may explain the abnormal cellular polarity and inability to secrete zymogen granules in pancreatic acinar exocrine cells. The nervous system appeared to develop normally, despite abundant expression of BETA2 in differentiating neurons. Thus, BETA2 is critical for the normal development of several specialized cell types arising from the gut endoderm.
DNA methylation inhibits elongation but not initiation of transcription in Neurospora crassa
Authors: Rountree Michael R. , Selker Eric U.
Published: February 2008

In plants, animals, and fungi, DNA methylation is frequently associated with gene silencing, yet little is known about the role of the methylation in silencing. In Neurospora crassa, repeated sequences are silenced by repeat-induced point mutation (RIP) and genes that have suffered numerous GC → AT mutations by RIP are typically methylated at remaining cytosines. We investigated possible effects on transcription from methylation associated with RIP by taking advantage of 5-azacytidine, which prevents most methylation in Neurosporaand a dim-2 mutation that abolishes all detectable methylation. Northern analyses revealed that methylation prevents the accumulation of transcripts from genes mutated by RIP. Measurements of transcription rates in vivo showed that methylation inhibits transcription severely but does not influence mRNA stability. Results of nuclear run-on experiments demonstrated that transcription initiation was not significantly inhibited by the dense methylation in the promoter sequences. In contrast, methylation blocked transcription elongation in vivo.
Rpp1, an essential protein subunit of nuclear RNase P required for processing of precursor tRNA and 35S precursor rRNA inSaccharomyces cerevisiae
Authors: Stolc Viktor , Altman Sidney
Published: February 2008

The gene for an essential protein subunit of nuclear RNase P fromSaccharomyces cerevisiae has been cloned. The gene for this protein, RPP1, was identified by virtue of its homology with a human scleroderma autoimmune antigen, Rpp30, which copurifies with human RNase P. Epitope-tagged Rpp1 can be found in association with both RNase P RNA and a related endoribonuclease, RNase MRP RNA, in immunoprecipitates from crude extracts of cells. Depletion of Rpp1 in vivo leads to the accumulation of precursor tRNAs with unprocessed 5′ and 3′ termini and reveals rRNA processing defects that have not been described previously for proteins associated with RNase P or RNase MRP. Immunoprecipitated complexes cleave both yeast precursor tRNAs and precursor rRNAs.
Mismatch repair protein MutL becomes limiting during stationary-phase mutation
Authors: Harris Reuben S. , Feng Gang , Ross Kimberly J. , Sidhu Roger , Thulin Carl , Longerich Simonne , Szigety Susan K. , Winkler Malcolm E. , Rosenberg Susan M.
Published: February 2008

Postsynthesis mismatch repair is an important contributor to mutation avoidance and genomic stability in bacteria, yeast, and humans. Regulation of its activity would allow organisms to regulate their ability to evolve. That mismatch repair might be down-regulated in stationary-phase Escherichia coli was suggested by the sequence spectrum of some stationary-phase (“adaptive”) mutations and by the observations that MutS and MutH levels decline during stationary phase. We report that overproduction of MutL inhibits mutation in stationary phase but not during growth. MutS overproduction has no such effect, and MutL overproduction does not prevent stationary-phase decline of either MutS or MutH. These results imply that MutS and MutH decline to levels appropriate for the decreased DNA synthesis in stationary phase, whereas functional MutL is limiting for mismatch repair specifically during stationary phase. Modulation of mutation rate and genetic stability in response to environmental or developmental cues, such as stationary phase and stress, could be important in evolution, development, microbial pathogenicity, and the origins of cancer.
K-ras is an essential gene in the mouse with partial functional overlap with N-ras
Authors: Johnson Leisa , Greenbaum Doron , Cichowski Karen , Mercer Kim , Murphy Elizabeth , Schmitt Eric , Bronson Roderick T. , Umanoff Heywood , Edelmann Windfried , Kucherlapati Raju , Jacks Tyler
Published: February 2008

Mammalian ras genes are thought to be critical in the regulation of cellular proliferation and differentiation and are mutated in ∼30% of all human tumors. However, N-ras and H-ras are nonessential for mouse development. To characterize the normal role of K-ras in growth and development, we have mutated it by gene targeting in the mouse. On an inbred genetic background, embryos homozygous for this mutation die between 12 and 14 days of gestation, with fetal liver defects and evidence of anemia. Thus, K-ras is the only member of the ras gene family essential for mouse embryogenesis. We have also investigated the effect of multiple mutations within the ras gene family. Most animals lacking N-ras function and heterozygous for the K-rasmutation exhibit abnormal hematopoietic development and die between days 10 and 12 of embryogenesis. Thus, partial functional overlap appears to occur within the ras gene family, but K-rasprovides a unique and essential function.
A multifunctional DNA-binding protein that promotes the formation of serum response factor/homeodomain complexes: identity to TFII-I
Authors: Grueneberg Dorre A. , Henry R. William , Brauer Andrew , Novina Carl D. , Cheriyath Venugopalan , Roy Ananda L. , Gilman Michael
Published: February 2008

The human homeodomain protein Phox1 interacts functionally with serum response factor (SRF) to impart serum responsive transcriptional activity to SRF-binding sites in a HeLa cell cotransfection assay. However, stable ternary complexes composed of SRF, Phox1, and DNA, which presumably mediate the transcriptional effects of Phox1 in vivo, have not been observed in vitro. Here, we report the identification, purification, and molecular cloning of a human protein that promotes the formation of stable higher-order complexes of SRF and Phox1. We show that this protein, termed SPIN, interacts with SRF and Phox1 in vitro and in vivo. Furthermore, SPIN binds specifically to multiple sequences in the c-fos promoter and interacts cooperatively with Phox1 to promote serum-inducible transcription of a reporter gene driven by the c-fos serum response element (SRE). SPIN is identical to the initiator-binding protein TFII-I. Consistent with this hypothesis, SPIN exhibits modest affinity for a characterized initiator sequence in vitro. We propose that this multifunctional protein coordinates the formation of an active promoter complex at the c-fos gene, including the linkage of specific signal responsive activator complexes to the general transcription machinery.
Translational repressorbruno plays multiple roles in development and is widely conserved
Authors: Webster Philippa J. , Liang Lu , Berg Celeste A. , Lasko Paul , Macdonald Paul M.
Published: February 2008

oskar (osk) mRNA is tightly localized to the posterior pole of the Drosophila oocyte, where the subsequent expression of Osk protein directs abdomen and germ-line formation in the developing embryo. Misplaced expression of Osk protein leads to lethal body patterning defects. The Osk message is translationally repressed before and during the localization process, ensuring that Osk protein is only expressed after the mRNA has reached the posterior. An ovarian protein, Bruno (Bru), has been implicated as a translational repressor of osk mRNA. Here we report the isolation of a cDNA encoding Bru using a novel approach to the expression cloning of an RNA-binding protein, and the identification of previously described mutants in the arrest (aret)-locus as mutants in Bru. The mutant phenotype, along with the binding properties of the protein and its pattern of accumulation within the oocyte, indicate that Bru regulates multiple mRNAs involved in female and male gametogenesis as well as early in embryogenesis. Genetic experiments provide further evidence that Bru functions in the translational repression of osk. Intriguingly, we find that Bru interacts physically with Vasa (Vas), an RNA helicase that is a positive regulator of osk translation. Bru belongs to an evolutionarily conserved family of genes, suggesting that Bru-mediated translational regulation may be widespread. Models for the molecular mechanism of Bru function are discussed.
Transcription elongation factor P-TEFb is required for HIV-1 Tat transactivation in vitro
Authors: Zhu Yuerong , Pe’ery Tsafrira , Peng Junmin , Ramanathan Yegnanarayana , Marshall Nick , Marshall Tricia , Amendt Brad , Mathews Michael B. , Price David H.
Published: February 2008

P-TEFb is a key regulator of the process controlling the processivity of RNA polymerase II and possesses a kinase activity that can phosphorylate the carboxy-terminal domain of the largest subunit of RNA polymerase II. Here we report the cloning of the small subunit ofDrosophila P-TEFb and the finding that it encodes a Cdc2-related protein kinase. Sequence comparison suggests that a protein with 72% identity, PITALRE, could be the human homolog of theDrosophila protein. Functional homology was suggested by transcriptional analysis of an RNA polymerase II promoter with HeLa nuclear extract depleted of PITALRE. Because the depleted extract lost the ability to produce long DRB-sensitive transcripts and this loss was reversed by the addition of purified Drosophila P-TEFb, we propose that PITALRE is a component of human P-TEFb. In addition, we found that PITALRE associated with the activation domain of HIV-1 Tat, indicating that P-TEFb is a Tat-associated kinase (TAK). An in vitro transcription assay demonstrates that the effect of Tat on transcription elongation requires P-TEFb and suggests that the enhancement of transcriptional processivity by Tat is attributable to enhanced function of P-TEFb on the HIV-1 LTR.
Mammalian homologs of seven in absentia regulate DCC via the ubiquitin–proteasome pathway
Authors: Hu Gang , Zhang Sheng , Vidal Marc , Baer Joshua La , Xu Tian , Fearon Eric R.
Published: February 2008

DCC (deleted in colorectalcancer) is postulated to function as transmembrane receptor for the axon and cell guidance factor netrin-1. We report here that the DCC cytoplasmic domain binds to proteins encoded by mammalian homologs of the Drosophila seven in absentia (sina) gene, as well as Drosophila Sina. Sina has a critical role in R7 photoreceptor development and shows upward of 85% amino acid identity with its mammalian homologs (termed Siahs), but the function of the Sina/Siah proteins has not been defined. We sought, therefore, to characterize further their interaction with DCC. Immunofluorescence studies suggested the Sina/Siah proteins localized predominantly in the cytoplasm and in association with DCC. DCC was found to be ubiquitinated and the Sina/Siah proteins regulated its expression. Proteasome inhibitors blocked the effects of Sina/Siah on DCC, and the Sina/Siah proteins interacted with ubiquitin-conjugating enzymes (Ubcs). A mutant Siah protein lacking the amino-terminal Ubc-binding sequences complexed with DCC, but did not degrade it. The in vivo interaction between Sina/Siah and DCC was confirmed through studies of transgenic Drosophilalines in which DCC and Sina were ectopically expressed in the eye. Taken together, the data imply that the Sina/Siah proteins regulate DCC and perhaps other proteins via the ubiquitin–proteasome pathway.
Casein kinase II regulation of yeast TFIIIB is mediated by the TATA-binding protein
Authors: Ghavidel Ataollah , Schultz Michael C.
Published: February 2008

The highly conserved protein kinase casein kinase II (CKII) is required for efficient Pol III transcription of the tRNA and 5S rRNA genes in Saccharomyces cerevisiae. Using purified factors from wild-type cells to complement transcription extracts from a conditional lethal mutant of CKII we show that TFIIIB is the CKII-responsive component of the Pol III transcription machinery. Dephosphorylation of TFIIIB eliminated its ability to complement CKII-depleted extract, and a single TFIIIB subunit, the TATA-binding protein (TBP), is a preferred substrate of CKII in vitro. Recombinant TBP purified fromEscherichia coli is phosphorylated efficiently by CKII and, in the presence of a limiting amount of CKII, is able to substantially rescue transcription in CKII-deficient extract. Our results establish that TBP is a key component of the pathway linking CKII activity and Pol III transcription and suggest that TBP is the target of a CKII-mediated regulatory mechanism that can modulate Pol III transcription.
The downstream core promoter element, DPE, is conserved fromDrosophila to humans and is recognized by TAFII60 of Drosophila
Authors: Burke Thomas W. , Kadonaga James T.
Published: February 2008

We analyzed the function of the downstream promoter element (DPE), a distinct 7-nucleotide core promoter element that is ∼30 nucleotides downstream of the transcription start site of many TATA-box-deficient (TATA-less) promoters in Drosophila. There is a strict requirement for spacing between the Inr and DPE motifs, as an increase or decrease of 3 nucleotides in the distance between the Inr and DPE causes a seven- to eightfold reduction in transcription as well as a significant reduction in the binding of purified TFIID. These results suggest a specific and somewhat rigid interaction of TFIID with the Inr and DPE sequences. Photo-cross-linking analysis of purified TFIID with a TATA-less DPE-containing promoter revealed specific cross-linking of dTAFII60 and dTAFII40 to the DPE, with a higher efficiency of cross-linking to dTAFII60 than to dTAFII40. These data, combined with the previously well-characterized interactions between the two TAFs and their homology to histones H4 and H3, suggest that a dTAFII60–dTAFII40 heterotetramer binds to the DPE. Human and Drosophila transcription factors exhibit essentially the same requirements for DPE sequence and for Inr–DPE spacing. In addition, the TATA-less promoter of the human interferon regulatory factor-1 (IRF-1) gene contains a DPE that is important for transcriptional activity both in vitro and in cultured cells. Hence, these studies provide evidence for a direct role of TAFs in basal transcription of TATA-less DPE-containing genes and collectively indicate that the DPE is, in many respects, a downstream counterpart to the TATA box that is present in Drosophila to humans.
Mouse mutant embryos overexpressing IGF-II exhibit phenotypic features of the Beckwith–Wiedemann and Simpson–Golabi–Behmel syndromes
Authors: Eggenschwiler Jonathan , Ludwig Thomas , Fisher Peter , Leighton Philip A. , Tilghman Shirley M. , Efstratiadis Argiris
Published: February 2008

In mice, the imprintedIgf2gene (expressed from the paternal allele), which encodes a growth-promoting factor (IGF-II), is linked closely to the reciprocally imprintedH19locus on chromosome 7. Also imprinted (expressed from the maternal allele) is theIgf2rgene on chromsome 17 encoding the type 2 IGF receptor that is involved in degradation of excess IGF-II. Double mutant embryos carrying a deletion around theH19region and also a targetedIgf2rallele, both inherited maternally, have extremely high levels of IGF-II (7- and 11-fold higher than normal in tissues and serum, respectively) as a result of biallelicIgf2expression (imprint relaxation by deletion ofH19-associated sequence) in combination with lack of the IGF2R-mediated IGF-II turnover. This excess of IGF-II causes somatic overgrowth, visceromegaly, placentomegaly, omphalocele, and cardiac and adrenal defects, which are also features of the Beckwith–Wiedemann syndrome (BWS), a genetically complex human disorder associated with chromosomal abnormalities in the 11p15.5 region where theIGF2gene resides. In addition, the double mutant mouse embryos exhibit skeletal defects and cleft palate, which are manifestations observed frequently in the Simpson–Golabi–Behmel syndrome, another overgrowth disorder overlapping phenotypically, but not genetically, with BWS.
sel-10, a negative regulator of lin-12 activity inCaenorhabditis elegans, encodes a member of the CDC4 family of proteins
Authors: Hubbard E. Jane Albert , Wu Guangyu , Kitajewski Jan , Greenwald Iva
Published: February 2008

Mutations that influence lin-12 activity inCaenorhabditis elegans may identify conserved factors that regulate the activity of lin-12/Notch proteins. We describe genetic evidence indicating that sel-10 is a negative regulator of lin-12/Notch-mediated signaling in C. elegans. Sequence analysis shows that SEL-10 is a member of the CDC4 family of proteins and has a potential human ortholog. Coimmunoprecipitation data indicate that C. elegansSEL-10 complexes with LIN-12 and with murine Notch4. We propose that SEL-10 promotes the ubiquitin-mediated turnover of LIN-12/Notch proteins, and discuss potential roles for the regulation of lin-12/Notch activity bysel-10 in cell fate decisions and tumorigenesis.
A novel cytosolic regulator, Pianissimo, is required for chemoattractant receptor and G protein-mediated activation of the 12 transmembrane domain adenylyl cyclase in Dictyostelium
Authors: Chen Mei-Yu , Long Yu , Devreotes Peter N.
Published: February 2008

Genetic analysis was applied to identify novel genes involved in G protein-linked pathways controlling development. Using restriction enzyme-mediated integration (REMI), we have identified a new gene,Pianissimo (PiaA), involved in cAMP signaling inDictyostelium discoideum. PiaA encodes a 130-kD cytosolic protein required for chemoattractant receptor and G protein-mediated activation of the 12 transmembrane domain adenylyl cyclase. InpiaA− null mutants, neither chemoattractant stimulation of intact cells nor GTPγS treatment of lysates activates the enzyme; constitutive expression of PiaA reverses these defects. Cytosols of wild-type cells that contain Pia protein reconstitute the GTPγS stimulation of adenylyl cyclase activity in piaA−lysates, indicating that Pia is directly involved in the activation. Pia and CRAC, a previously identified cytosolic regulator, are both essential for activation of the enzyme as lysates of crac−piaA− double mutants require both proteins for reconstitution. Homologs of PiaA are found in Saccharomyces cerevisiaeand Schizosaccaromyces pombe; disruption of the S. cerevisiae homolog results in lethality. We propose that homologs of Pia and similar modes of regulation of these ubiquitous G protein-linked pathways are likely to exist in higher eukaryotes.
Histone acetyltransferases regulate HIV-1 enhancer activity in vitro
Authors: Sheridan Philip L. , Mayall Timothy P. , Verdin Eric , Jones Katherine A.
Published: February 2008

Specific inhibitors of histone deacetylase, such as trichostatin A (TSA) and trapoxin (TPX), are potent inducers of HIV-1 transcription in latently infected T-cell lines. Activation of the integrated HIV-1 promoter is accompanied by the loss or rearrangement of a positioned nucleosome (nuc-1) near the viral RNA start site. Here we show that TSA strongly induces HIV-1 transcription on chromatin in vitro, concomitant with an enhancer factor-assisted increase in the level of acetylated histone H4. TSA treatment, however, did not detectably alter enhancer factor binding or the positioning of nuc-1 on the majority of the chromatin templates indicating that protein acetylation and chromatin remodeling may be limiting steps that occur only on transcriptionally competent templates, or that remodeling of nuc-1 requires additional factors. To assess the number of active chromatin templates in vitro, transcription was limited to a single round with low levels of the detergent Sarkosyl. Remarkably, HIV-1 transcription on chromatin was found to arise from a small number of active templates that can each support nearly 100 rounds of transcription, and TSA increased the number of active templates in each round. In contrast, transcription on naked DNA was limited to only a few rounds and was not responsive to TSA. We conclude that HIV-1 enhancer complexes greatly facilitate transcription reinitiation on chromatin in vitro, and act at a limiting step to promote the acetylation of histones or other transcription factors required for HIV-1 enhancer activity.
DNA damage induces phosphorylation of the amino terminus of p53
Authors: Siliciano Janet D. , Canman Christine E. , Taya Yoichi , Sakaguchi Kazuyasu , Appella Ettore , Kastan Michael B.
Published: February 2008

Data are presented demonstrating that DNA damage leads to specific post-translational modifications of p53 protein. Using two-dimensional peptide mapping of in vivo radiolabeled p53 tryptic phosphopeptides, recombinant truncated p53 protein, and synthetic p53 tryptic peptides, a unique p53 phosphopeptide was identified after exposure of ML-1 cells to ionizing irradiation. This peptide represents the first 24 amino acids of p53 and contains three phosphorylated serine residues. A specific p53 phosphopeptide antibody identified serine-15 as one of the two serines in p53 that becomes phosphorylated following DNA damage induced by either ionizing irradiation (IR) or ultraviolet (UV) irradiation in multiple cell types. IR-induced phosphorylation of p53 does not affect the kinetics of p53 binding to or dissociating from DNA as assessed by electrophoretic mobility-shift assays. However, p53 phosphorylation induced by DNA damage correlates with enhanced transcription of downstream p53 target genes. Low levels of phosphoserine-15 p53 are detectable within 6 hr after IR in AT cells, whereas lymphoblasts from normal individuals exhibit this modification within 1 hr. In contrast, phosphorylation of p53 on serine-15 is similar in normal and AT cells after UV irradiation. Our results indicate that p53 is phosphorylated in response to DNA damage, that this de novo phosphorylation may be involved in the subsequent induction and activation of p53, and that although ATM affects the kinetics of p53 phosphorylation after IR, it is not absolutely required for phosphorylation of p53 on serine-15.
Requirement for NF-κB in osteoclast and B-cell development
Authors: Franzoso Guido , Carlson Louise , Xing Lianping , Poljak Ljiljana , Shores Elizabeth W. , Brown Keith D. , Leonardi Antonio , Tran Tom , Boyce Brendan F. , Siebenlist Ulrich
Published: February 2008

NF-κB is a family of related, dimeric transcription factors that are readily activated in cells by signals associated with stress or pathogens. These factors are critical to host defense, as demonstrated previously with mice deficient in individual subunits of NF-κB. We have generated mice deficient in both the p50 and p52 subunits of NF-κB to reveal critical functions that may be shared by these two highly homologous proteins. We now demonstrate that unlike the respective single knockout mice, the p50/p52 double knockout mice fail to generate mature osteoclasts and B cells, apparently because of defects that track with these lineages in adoptive transfer experiments. Furthermore, these mice present markedly impaired thymic and splenic architectures and impaired macrophage functions. The blocks in osteoclast and B-cell maturation were unexpected. Lack of mature osteoclasts caused severe osteopetrosis, a family of diseases characterized by impaired osteoclastic bone resorption. These findings now establish critical roles for NF-κB in development and expand its repertoire of roles in the physiology of differentiated hematopoietic cells.
A prokaryotic-like mode of cytoplasmic eukaryotic ribosome binding to the initiation codon during internal translation initation of hepatitis C and classical swine fever virus RNAs
Authors: Pestova Tatyana V. , Shatsky Ivan N. , Fletcher Simon P. , Jackson Richard J. , Hellen Christopher U.T.
Published: February 2008

Initiation of translation of hepatitis C virus and classical swine fever virus mRNAs results from internal ribosomal entry. We reconstituted internal ribosomal entry in vitro from purified translation components and monitored assembly of 48S ribosomal preinitiation complexes by toe-printing. Ribosomal subunits (40S) formed stable binary complexes on both mRNAs. The complex structure of these RNAs determined the correct positioning of the initiation codon in the ribosomal “P” site in binary complexes. Ribosomal binding and positioning on these mRNAs did not require the initiation factors eIF3, eIF4A, eIF4B, and eIF4F and translation of these mRNAs was not inhibited by a trans-dominant eIF4A mutant. Addition of Met–tRNAiMet, eIF2, and GTP to these binary ribosomal complexes resulted in formation of 48S preinitiation complexes. The striking similarities between this eukaryotic initiation mechanism and the mechanism of translation initiation in prokaryotes are discussed.
Asymmetric segregation on spindle poles of theSchizosaccharomyces pombeseptum-inducing protein kinase Cdc7p
Authors: Sohrmann Marc , Schmidt Susanne , Hagan Iain , Simanis Viesturs
Published: February 2008

Schizosaccharomyces pombedivides by means of a centrally placed division septum. The initiation of septation must be tightly coordinated with events in mitosis, as premature formation of the septum can lethally cut the undivided nucleus. The Spg1p GTPase and the Cdc7p kinase, with which it interacts, play a central role in signaling the initiation of septum formation. Loss-of-function mutations in either gene prevent septation, whereas inappropriate activation of Spg1p can induce septum formation from G1or G2interphase cells. Increased expression of either gene leads to multiple rounds of septation without cell cleavage, emphasizing the need for precise cell cycle regulation of their activity. To understand the mechanisms underlying this regulation, we have investigated whether these key initiators of septum formation are controlled by changes in their activity and/or location during mitosis and cytokinesis. We demonstrate that Spg1p localizes to the spindle pole body in interphase and to both spindle poles during mitosis. In contrast, Cdc7p shows no discrete localization during interphase, but early in mitosis it associates with both spindle pole bodies and, as the spindle extends, is seen on only one pole of the spindle during anaphase B. Spg1p activity is required for localization of Cdc7p in vivo but not for its kinase activity in vitro. Staining with an antiserum that recognizes preferentially GDP–Spg1p indicates that activated GTP–Spg1p predominates during mitosis when Cdc7p is associated with the spindle pole body. Furthermore, staining with this antibody shows that asymmetric distribution of Cdc7p may be mediated by inactivation of Spg1p on one spindle pole. Deregulated septation in mutant cells correlates with segregation of Cdc7p to both spindle poles.
Regulation of the fission yeast transcription factor Pap1 by oxidative stress: requirement for the nuclear export factor Crm1 (Exportin) and the stress-activated MAP kinase Sty1/Spc1
Authors: Toone W. Mark , Kuge Shusuke , Samuels Michael , Morgan Brian A. , Toda Takashi , Jones Nic
Published: February 2008

The fission yeast Sty1 stress-activated MAP kinase is crucial for the cellular response to a variety of stress conditions. Accordingly,sty1− cells are defective in their response to nutrient limitation, lose viability in stationary phase, and are hypersensitive to osmotic stress, oxidative stress, and UV treatment. Some of these phenotypes are caused by Sty1-dependent regulation of the Atf1 transcription factor, which controls both meiosis-specific and osmotic stress-responsive genes. However, in this report we demonstrate that the cellular response to oxidative stress and to treatment with a variety of cytotoxic agents is the result of Sty1 regulation of the Pap1 transcription factor, a bZip protein with structural and DNA binding similarities to the mammalian c-Jun protein. We show that both Sty1 and Pap1 are required for the expression of a number of genes involved in the oxidative stress response and for the expression of two genes, hba2+/bfr1+ andpmd1+, which encode energy-dependent transport proteins involved in multidrug resistance. Furthermore, we demonstrate that Pap1 is regulated by stress-dependent changes in subcellular localization. On imposition of oxidative stress, the Pap1 protein relocalizes from the cytoplasm to the nucleus in a process that is dependent on the Sty1 kinase. This relocalization is the result of regulated protein export, rather than import, and involves the Crm1 (exportin) nuclear export factor and the dcd1+/pim1+ gene that encodes an Ran nucleotide exchange factor.
The histone acetylase PCAF is a nuclear receptor coactivator
Authors: Blanco Jorge C.G. , Minucci Saverio , Lu Jianming , Yang Xiang-Jiao , Walker Kristen K. , Chen Hongwu , Evans Ronald M. , Nakatani Yoshihiro , Ozato Keiko
Published: February 2008

Whereas the histone acetylase PCAF has been suggested to be part of a coactivator complex mediating transcriptional activation by the nuclear hormone receptors, the physical and functional interactions between nuclear receptors and PCAF have remained unclear. Our efforts to clarify these relationships have revealed two novel properties of nuclear receptors. First, we demonstrate that the RXR/RAR heterodimer directly recruits PCAF from mammalian cell extracts in a ligand-dependent manner and that increased expression of PCAF leads to enhanced retinoid-responsive transcription. Second, we demonstrate that, in vitro, PCAF directly associates with the DNA-binding domain of nuclear receptors, independently of p300/CBP binding, therefore defining a novel cofactor interaction surface. Furthermore, our results show that dissociation of corepressors enables ligand-dependent PCAF binding to the receptors. This observation illuminates how a ligand-dependent receptor function can be propagated to regions outside the ligand-binding domain itself. On the basis of these observations, we suggest that PCAF may play a more central role in nuclear receptor function than previously anticipated.
The surveillance complex interacts with the translation release factors to enhance termination and degrade aberrant mRNAs
Authors: Czaplinski Kevin , Ruiz-Echevarria Maria J. , Paushkin Sergey V. , Han Xia , Weng Youmin , Perlick Haley A. , Dietz Harry C. , Ter-Avanesyan Michael D. , Peltz Stuart W.
Published: February 2008

The nonsense-mediated mRNA decay pathway is an example of an evolutionarily conserved surveillance pathway that rids the cell of transcripts that contain nonsense mutations. The product of theUPF1 gene is a necessary component of the putative surveillance complex that recognizes and degrades aberrant mRNAs. Recent results indicate that the Upf1p also enhances translation termination at a nonsense codon. The results presented here demonstrate that the yeast and human forms of the Upf1p interact with both eukaryotic translation termination factors eRF1 and eRF3. Consistent with Upf1p interacting with the eRFs, the Upf1p is found in the prion-like aggregates that contain eRF1 and eRF3 observed in yeast [PSI+] strains. These results suggest that interaction of the Upf1p with the peptidyl release factors may be a key event in the assembly of the putative surveillance complex that enhances translation termination, monitors whether termination has occurred prematurely, and promotes degradation of aberrant transcripts.
The DNA methylation locusDDM1 is required for maintenance of gene silencing in Arabidopsis
Authors: Jeddeloh Jeffrey A. , Bender Judith , Richards Eric J.
Published: February 2008

To investigate the relationship between cytosine methylation and gene silencing in Arabidopsis, we constructed strains containing the ddm1 hypomethylation mutation and a methylated and silenced PAI2 tryptophan biosynthetic gene (MePAI2) that results in a blue fluorescent plant phenotype. The ddm1mutation had both an immediate and a progressive effect on PAIgene silencing. In the first generation, homozygous ddm1 MePAI2plants displayed a weakly fluorescent phenotype, in contrast to the strongly fluorescent phenotype of the DDM1 MePAI2 parent. After two generations of inbreeding by self-pollination, theddm1/ddm1 lines became nonfluorescent. The progressive loss of fluorescence correlated with a progressive loss of methylation from the PAI2 gene. These results indicate that methylation is necessary for maintenance of PAI gene silencing and that intermediate levels of DNA methylation are associated with intermediate gene silencing. The results also support our earlier hypothesis that ddm1 homozygotes act as “epigenetic mutators” by accumulating heritable changes in DNA methylation that can lead to changes in gene expression.
Myc activates telomerase
Authors: Wang Jing , Xie Lin Ying , Allan Susan , Beach David , Hannon Gregory J.
Published: February 2008

Telomere maintenance has been proposed as an essential prerequisite to human tumor development. The telomerase enzyme is itself a marker for tumor cells, but the genetic alterations that activate the enzyme during neoplastic transformation have remained a mystery. Here, we show that Myc induces telomerase in both normal human mammary epithelial cells (HMECs) and normal human diploid fibroblasts. Myc increases expression of hEST2 (hTRT/TP2), the limiting subunit of telomerase, and both Myc and hEST2 can extend the life span of HMECs. The ability of Myc to activate telomerase may contribute to its ability to promote tumor formation.
Seven-up, the Drosophila homolog of the COUP-TF orphan receptors, controls cell proliferation in the insect kidney
Authors: Kerber Birgit , Fellert Sonja , Hoch Michael
Published: February 2008

Morphogenesis of the insect kidney, the Malpighian tubules, is controlled in Drosophila by a single large cell, the tip cell. It has been postulated that this cell sends out a mitogenic signal that induces the division of neighboring cells. The signal and the molecules that receive it have remained elusive. We show that the COUP-TF-related nuclear orphan receptor Seven-up is a key component that becomes induced in response to mitogenic EGF receptor signaling activity emanating from the tip cell. Seven-up in turn is capable of regulating the transcription of cell cycle regulators.