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PubMed2005

Characterization of functional domains of the Vibrio cholerae virulence regulator ToxT.

The toxT gene encodes an AraC family transcriptional activator that is responsible for regulating virulence gene expression in Vibrio cholerae. Analysis of ToxT by dominant/negative assays and a LexA-based reporter system demonstrated that the N-terminus of the protein contains dimerization determinants, indicating that ToxT likely functions as a dimer. Additionally, a natural variant of ToxT with only 60% identity in the N-terminus, as well as a mutant form of ToxT with an altered amino acid in the N-terminus (L107F), exhibited altered transcriptional responses to bile, suggesting that the N-terminus is involved in environmental sensing. The C-terminus of ToxT functions to bind DNA and requires dimerization for stable binding in vitro, as demonstrated by gel shift analysis. Interestingly, a dimerized form of the ToxT C-terminus is able to bind DNA in vitro but is transcriptionally inactive in vivo, indicating that the N-terminus contains determinants that are required for transcriptional activation. These results provide a model for a two-domain structure for ToxT, with an N-terminal dimerization and environmental sensing domain and a C-terminal DNA-binding domain; unlike other well-studied AraC family proteins, both domains of ToxT appear to be required for transcriptional activation.

Amino Acid Substitution↗
PubMed1989

EBV gene expression in an NPC-related tumour.

A nasopharyngeal carcinoma tumour (designated C15) propagated in nude mice has been used to generate a large cDNA library that we have analysed for Epstein-Barr virus (EBV) gene expression. No gross alterations exist in viral DNA from C15 relative to other human isolates and the large deletion present in the B95-8 'prototype' viral strain established in marmoset cells is not found; C15 contains no linear virion DNA. In the cDNA library, of the six EBV nuclear antigens (EBNAs) expressed in latently infected B-lymphocytes, only clones for EBNA-1 are found. These data are confirmed by immunoblotting. Sequence analysis shows the EBNA-1 mRNA splicing pattern in the carcinoma to differ from that observed in B-lymphocytes. Further, contrary to observations with B-cell lines, most viral transcription in the tumour is localized onto the 'rightmost' region of the conventional EBV physical map. Transcripts identified corresponding to known genes include those for the latent membrane protein (LMP), the alkaline DNA exonuclease and probably the terminal protein; major transcripts are also derived from the BamHI D fragment and the region deleted in B95-8 EBV DNA. Novel transcripts have also been identified that proceed in an anti-sense direction to genes encoding functions associated with replication, such as the viral DNA polymerase. They contain a large, hitherto unidentified, open reading frame in the viral genome that is complementary to the putative function known as BALF3 and a smaller open reading frame complementary to BALF5 (the DNA polymerase gene). From the present studies we can conclude that: (i) EBV transcription patterns in the epithelial cells vary markedly from those identified previously in B-cells, reflecting differential use of promoters or splicing patterns. (ii) Transcription is tightly regulated and restricted in the C15 tumour with many latent genes, notably EBNAs 2-6, being 'switched off.' (iii) A family of cytoplasmic RNAs are transcribed in an antisense direction to a number of existing open reading frames in the EBV genome. (iv) There are a number of mutations in C15 transcripts relative to the B95-8 genome, some of which could result in amino acid alterations in proteins.

Amino Acid Sequence↗
PubMed2005

Construction, analysis, and beta-glucanase screening of a bacterial artificial chromosome library from the large-bowel microbiota of mice.

A metagenomic (community genomic) library consisting of 5,760 bacterial artificial chromosome clones was prepared in Escherichia coli DH10B from DNA extracted from the large-bowel microbiota of BALB/c mice. DNA inserts detected in 61 randomly chosen clones averaged 55 kbp (range, 8 to 150 kbp) in size. A functional screen of the library for beta-glucanase activity was conducted using lichenin agar plates and Congo red solution. Three clones with beta-glucanase activity were detected. The inserts of these three clones were sequenced and annotated. Open reading frames (ORF) that encoded putative proteins with identity to glucanolytic enzymes (lichenases and laminarinases) were detected by reference to databases. Other putative genes were detected, some of which might have a role in environmental sensing, nutrient acquisition, or coaggregation. The insert DNA from two clones probably originated from uncultivated bacteria because the ORF had low sequence identity with database entries, but the genes associated with the remaining clone resembled sequences reported in Bacteroides species.

Amino Acid Sequence↗
PubMed2002

An imprinted PEG1/MEST antisense expressed predominantly in human testis and in mature spermatozoa.

PEG1 (or MEST) is an imprinted gene located on human chromosome 7q32 that is expressed predominantly from the paternal allele. In the mouse, Peg1/Mest is associated with embryonic growth and maternal behavior. Human PEG1 is transcribed from two promoters; the transcript from promoter P1 is derived from both parental alleles, and the transcript from P2 is exclusively from the paternal allele. We characterized the P1 and P2 transcripts in various normal and neoplastic tissues. In the normal tissues, PEG1 was transcribed from both promoters P1 and P2, whereas in six of eight neoplastic tissues, PEG1 was transcribed exclusively from promoter P1. Bisulfite sequencing demonstrated high levels of CpG methylation in the P2 region of DNA from a lung tumor. In the region between P1 and P2, we identified a novel transcript, PEG1-AS, in an antisense orientation to PEG1. PEG1-AS is a spliced transcript and was detected as a strong 2.4-kilobase band on a Northern blot. PEG1-AS and PEG1 P2-sense transcript were expressed exclusively from the paternal allele. Fragments of DNA from within the 1.5-kilobase region between PEG1-AS and the P2 exon were ligated to a pGL3 luciferase reporter vector and transfected into NCI H23 cells. This DNA exhibited strong promoter activity in both the sense and antisense directions, indicating that PEG1-AS and P2 exon share a common promoter region. Treatment of the transfected DNA fragments with CpG methylase abolished the promoter activity. Of interest, PEG1-AS was expressed predominantly in testis and in mature motile spermatozoa, indicating a possible role for this transcript in human sperm physiology and fertilization.

Alleles↗
PubMedDate not supplied

Electrostatic binding with tat and other cationic peptides increases cell accumulation of 99mTc-antisense DNAs without entrapment.

UNLABELLED: An important limitation restricting antisense nuclear medicine imaging is low radioactivity accumulations in target cells. The Tat peptide (Tat), a basic domain of the HIV Tat protein, has been shown to enhance cell accumulation of various biomolecules. PURPOSE: The influence of Tat, as a cationic carrier, on the accumulation in cell culture of anionic antisense DNAs bound electrostatically rather than covalently was investigated. To establish specificity of the accumulation, antisense DNA and control sequence were studied along with four different peptides. The technique of in situ reverse transcription was used to assay the in vivo hybridization of antisense DNA to the target mRNA in cultured live cells when transducted with the Tat peptide. METHODS: Uniform phosphorothioated DNAs were radiolabeled with 99mTc via Hynic/tricine. This 18 mer antisense DNA against RI alpha mRNA along with its sense and random control was studied in ACHN cells with the four peptides as carriers. RESULTS: The addition of Tat significantly increased cell accumulations. At 12 hours accumulations went from 14% to 45% for the antisense DNA and from 4% to 12% for control. Furthermore, an antisense effect was again suggested, now with the Tat carrier, by the significantly higher accumulation of 99mTc on both antisense DNAs vs. controls. Moreover, the accumulated antisense DNA enhanced with the Tat carrier was capable of priming reverse transcription as determined by an in situ assay suggesting that the DNA could escape from entrapment in endosome or lysosome vesicles for hybridization. However, differences in cellular accumulation with either Tat compared to either scrambled peptide were not significant, showing that the Tat in this study was functioning merely as a cationic carrier. CONCLUSIONS: Although electrostatic binding of antisense DNA to Tat is convenient, the association may mask the unique transduction properties of the peptide. Nevertheless, a promising improvement in cellular accumulation of antisense DNA was observed through the use of these carriers. In addition, at least a fraction of the transducted DNA appears to be free of entrapment to hybridize to its mRNA target in live cells.

DNA, Antisense↗
PubMed2005

ATM and ATR check in on origins: a dynamic model for origin selection and activation.

Initiation of DNA replication occurs at origins of replication, traditionally defined by specific sequence elements. Sequence-dependent initiation of replication is the rule in prokaryotes and in the yeast Saccharomyces cereviseae. However, sequence-dependent initiation does not appear to be absolutely required in metazoan eukaryotes. Origin firing is instead likely dependent on stochastic initiation from chromatin-defined loci, despite the demonstration of some specific origins. Based on some recent observations in Xenopus laevis egg extracts and in mammalian cell culture, we propose that timing of origin firing is dependent on feedback from active replicons. This dynamic regulation of replication is mediated by sensing of ongoing replication by the DNA-damage checkpoint kinases ATM and ATR, which in turn downregulate neighboring and distal origins and replicons by inhibition of the S-phase kinases Cdk2 and Cdc7 and by inhibition of the replicative Mcm helicase. Origin selection, activation, and replicon progression are therefore constrained in both space and time via feedback from the cell cycle and ongoing replication.

Animals↗
PubMed2003

Transgenic tobacco expressing geminiviral RNAs are resistant to the serious viral pathogen causing cotton leaf curl disease.

Cotton, the major cash crop in Pakistan, suffers 30% losses to cotton leaf curl disease, caused by the geminivirus, cotton leaf curl virus DNA A, plus a satellite component, DNA beta responsible for symptom development with plants failing to produce cotton bolls. We constructed transgenic tobacco expressing sense and antisense RNAs representing: [i] the 5' half of the viral DNA replication gene, AC1, [ii] the 3' half of AC1, [iii] two overlapping genes, AC2, a transcription activator, and AC3, a replication enhancer. In contrast to controls, 25% of 72 transgenic tobacco lines tested showed heritable resistance [T(1) - T(3) generations]: symptom-free and no replication of DNA A or DNA beta even after 120 days of continuous exposure to viruliferous whiteflies. As geminiviral and transgene RNAs are not detected in resistant lines following infection, and selected uninfected resistant tobacco sense lines reveal double-stranded and small interfering RNAs, the most likely mechanism is via post-transcriptional gene silencing.

DNA Replication↗
PubMed2003

[Mutations of fragile histidine triad gene in Peutz-Jeghers syndrome and canceration].

BACKGROUND & OBJECTIVE: Peutz-Jeghers syndrome (PJS) is an autosomal dominantly inherited disease. Fragile histidine triad (FHIT) gene is an important tumor suppressor gene at the fragile sites region of 3p14. The authors' previous study suggested that PJS patients might have a susceptible gene at the region of 3p14.2. This study was designed to reveal the relationship between the variant of FHIT gene in PJS and its canceration. METHOD: Mutations of FHIT gene in 15 PJS patients and 20 unaffected members in 6 PJS families were determined using denaturing high-performance liquid chromatography (DHPLC), polymerase chain reaction-single strand conformation polymorphism(PCR-SSCP) and DNA sequencing techniques. RESULTS: A non-sense mutation and a frame-shift mutation were identified at codon 54(GAA to TAA) (exon 6) which led to the change of the amino acid from glutamic acid (Glu) to stop codon, and a guanine insertion at codon 62 in exon 6 resulting in a premature stop codon TGA at codon 111 in one PJS patient. A homozygous deletion and a synonymous mutation were detected in exon 8. The homozygous deletion of exon 8 in FHIT gene was found in two polyps tissues and two cancerous tissues. And in 3 sporadic cases, the patients and their mothers have the same bands of SSCP and the same elution profiles of DHPLC when exon 8 was amplified. The DNA sequencing result showed that a synonymous mutation (polymorphism) occurred at codon 98 [CAT (H)-->CAC (H)], this mutation resulted in no change of amino acid. In addition, one base substitute from A to G mutation at 5'end, +42 nucleotide in intron 6 of FHIT gene was detected in seven patients and two unaffected members. CONCLUSION: PJS patients have low frequency point mutation of FHIT gene and their cancerous tissues had homozygous deletions in FHIT gene. This study indicated that the mutations and deletions of FHIT gene in PJS may play a role in the development of PJS and their cancerations.

Acid Anhydride Hydrolases↗
PubMed2006

Analyzing the dose-dependence of the Saccharomyces cerevisiae global transcriptional response to methyl methanesulfonate and ionizing radiation.

BACKGROUND: One of the most crucial tasks for a cell to ensure its long term survival is preserving the integrity of its genetic heritage via maintenance of DNA structure and sequence. While the DNA damage response in the yeast Saccharomyces cerevisiae, a model eukaryotic organism, has been extensively studied, much remains to be elucidated about how the organism senses and responds to different types and doses of DNA damage. We have measured the global transcriptional response of S. cerevisiae to multiple doses of two representative DNA damaging agents, methyl methanesulfonate (MMS) and gamma radiation. RESULTS: Hierarchical clustering of genes with a statistically significant change in transcription illustrated the differences in the cellular responses to MMS and gamma radiation. Overall, MMS produced a larger transcriptional response than gamma radiation, and many of the genes modulated in response to MMS are involved in protein and translational regulation. Several clusters of coregulated genes whose responses varied with DNA damaging agent dose were identified. Perhaps the most interesting cluster contained four genes exhibiting biphasic induction in response to MMS dose. All of the genes (DUN1, RNR2, RNR4, and HUG1) are involved in the Mec1p kinase pathway known to respond to MMS, presumably due to stalled DNA replication forks. The biphasic responses of these genes suggest that the pathway is induced at lower levels as MMS dose increases. The genes in this cluster with a threefold or greater transcriptional response to gamma radiation all showed an increased induction with increasing gamma radiation dosage. CONCLUSION: Analyzing genome-wide transcriptional changes to multiple doses of external stresses enabled the identification of cellular responses that are modulated by magnitude of the stress, providing insights into how a cell deals with genotoxicity.

DNA Damage↗
PubMed1998

Gene silencing from plant DNA carried by a Geminivirus.

The geminivirus tomato golden mosaic virus (TGMV) replicates in nuclei and expresses genes from high copy number DNA episomes. The authors used TGMV as a vector to determine whether episomal DNA can cause silencing of homologous, chromosomal genes. Two markers were used to asses silencing: (1) the sulfur allele (su) of magnesium chelatase, an enzyme required for chlorophyll formation; and (2) the firefly luciferase gene (luc). Various portions of both marker genes were inserted into TGMV in place of the coat protein open-reading frame and the constructs were introduced into intact plants using particle bombardment. When TGMV vectors carrying fragments of su (TGMV::su) were introduced into leaves of wild type Nicotiana benthamiana, circular, yellow spots with an area of several hundred cells formed after 3-5 days. Systemic movement of TGMV::su subsequently produced varigated leaf and stem tissue. Fragments that caused silencing included a 786 bp 5' fragment of the 1392 bp su cDNA in sense and anti-sense orientation, and a 403 bp 3' fragment. TGMV::su-induced silencing was propogated through tissue culture, along with the viral episome, but was not retained through meiosis. Systemic downregulation of a constitutively expresse luciferase transgene in plants was achieved following infection with TGMV vectors carrying a 623 bp portion of luc in sense or anti-sense orientation. These results establish that homologous DNA sequences localized in nuclear episomes can modulate the expression of active chromosomal genes.

Journal Article↗
PubMed1992

Gene conversion in the Escherichia coli RecF pathway: a successive half crossing-over model.

Gene conversion--apparently non-reciprocal transfer of sequence information between homologous DNA sequences--has been reported in various organisms. Frequent association of gene conversion with reciprocal exchange (crossing-over) of the flanking sequences in meiosis has formed the basis of the current view that gene conversion reflects events at the site of interaction during homologous recombination. In order to analyze mechanisms of gene conversion and homologous recombination in an Escherichia coli strain with an active RecF pathway (recBC sbcBC), we first established in cells of this strain a plasmid carrying two mutant neo genes, each deleted for a different gene segment, in inverted orientation. We then selected kanamycin-resistant plasmids that had reconstituted an intact neo+ gene by homologous recombination. We found that all the neo+ plasmids from these clones belonged to the gene-conversion type in the sense that they carried one neo+ gene and retained one of the mutant neo genes. This apparent gene conversion was, however, only very rarely accompanied by apparent crossing-over of the flanking sequences. This is in contrast to the case in a rec+ strain or in a strain with an active RecE pathway (recBC sbcA). Our further analyses, especially comparisons with apparent gene conversion in the rec+ strain, led us to propose a mechanism for this biased gene conversion. This "successive half crossing-over model" proposes that the elementary recombinational process is half crossing-over in the sense that it generates only one recombinant DNA duplex molecule, and leaves one or two free end(s), out of two parental DNA duplexes. The resulting free end is, the model assumes, recombinogenic and frequently engages in a second round of half crossing-over with the recombinant duplex. The products resulting from such interaction involving two molecules of the plasmid would be classified as belonging to the gene-conversion type without crossing-over. We constructed a dimeric molecule that mimics the intermediate form hypothesized in this model and introduced it into cells. Biased gene conversion products were obtained in this reconstruction experiment. The half crossing-over mechanism can also explain formation of huge linear multimers of bacterial plasmids, the nature of transcribable recombination products in bacterial conjugation, chromosomal gene conversion not accompanied by flanking exchange (like that in yeast mating-type switching), and antigenic variation in microorganisms.

Animals↗
PubMed1999

Antisense oligodeoxynucleotides downregulated c-sis mRNA expression to inhibit proliferation of vascular smooth muscle cells.

AIM: To assess the inhibitory effect of c-sis antisense oligodeoxynucleotides (ODN) on vascular smooth muscle cells (VSMC) proliferation and c-sis proto-oncogene mRNA expression. METHODS: The VSMC were cultured with Dulbecco's modified Engle's medium (DMEM) containing synthesized c-sis antisense or sense oligomers, [3H]thymidine (TdR) incorporation into DNA was determined by liquid scintillation counter and the numbers of cells were counted by cell counting plate, the mRNA level was observed by using of reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: C-sis antisense ODN 2, 4, 6, 8, and 10 mumol.L-1 inhibited VSMC proliferation (10.3% +/- 0.7%, 22.6% +/- 0.9%, 31.0% +/- 1.1%, 35.4% +/- 0.9%, and 43.3% +/- 1.2%) and [3H]TdR incorporation (6.8% +/- 0.3%, 9.7% +/- 0.7%, 29.0% +/- 0.6%, 32.0% +/- 0.7%, and 50.6% +/- 1.3%) in a concentration-dependent manner. When VSMC were treated with antisense ODN 10 mumol.L-1 for 4 d, the highest inhibitory rate of VSMC growth and that of [3H]TdR incorporation were 60.3% +/- 1.0% and 56.3% +/- 0.9%, respectively. RT-PCR showed that c-sis proto-oncogene antisense ODN downregulated c-sis mRNA expression obviously, while c-sis sense ODN did not inhibit VSMC proliferation and expression of c-sis proto-oncogene mRNA. CONCLUSION: The c-sis antisense ODN inhibited VSMC proliferation and downregulated c-sis mRNA level.

Animals↗
PubMed1998

Induction of microbial secondary metabolism.

Precursors often stimulate production of secondary metabolites either by increasing the amount of a limiting precursor, by inducing a biosynthetic enzyme (synthase) or both. These are usually amino acids but other small molecules also function as inducers. The most well-known are the auto-inducers which include gamma-butyrolactones (butanolides) of the actinomycetes, N-acylhomoserine lactones of Gram-negative bacteria, oligopeptides of Gram-positive bacteria, and B-factor (3'-[1-butylphosphoryl] adenosine) of Amycolatopsis mediterranei. The actinomycete butanolides exert their effects via receptor proteins which normally repress chemical and morphological differentiation (secondary metabolism and differentiation into aerial mycelia and spores respectively) but, when complexed with the butanolide, can no longer function. Homoserine lactones of Gram-negative bacteria function at high cell density and are structurally related to the butanolides. They turn on plant and animal virulence, light emission, plasmid transfer, and production of pigments, cyanide and beta-lactam antibiotics. They are made by enzymes homologous to Lux1, excreted by the cell, enter other cells at high density, bind to a LuxR homologue, the complex then binding to DNA upstream of genes controlled by "quorum sensing" and turning on their expression. Quorum sensing also operates in the case of the peptide pheromones of the Gram-positive bacteria. Here, secretion is accomplished by an ATP binding casette (ABC transporter), the secreted pheromone being recognized by a sensor component of a two-component signal transduction system. The pheromone often induces its own synthesis as well as those proteins involved in protein/peptide antibiotic (including bacteriocins and lantibiotics) production, virulence and genetic competence. The B-factor of A. mediterranei is an inducer of ansamycin (rifamycin) formation.

4-Butyrolactone↗
PubMed1998

The UL112/113 gene products of human cytomegalovirus which colocalize with viral DNA in infected cell nuclei are related to efficient viral DNA replication.

The UL112/113 gene products of human cytomegalovirus (HCMV) were shown by transient complementation ori Lyt-dependent DNA replication assay to be early viral proteins required for efficient viral DNA synthesis. By immunofluorescence analysis followed by fluorescence in situ hybridization, we showed that UL112/113 gene products of HCMV are colocalized with viral DNA prior to and during viral DNA replication in infected cell nuclei. We have used an anti-sense RNA approach for functional analysis of the UL112/113 gene in HCMV. The astrocytoma cell line U373-MG was used for permanent expression of the anti-sense UL112/113 gene. Expression of the anti-sense RNA in this cell line significantly blocked expression of UL112/113 gene products and viral DNA replication, indicating that the UL112/113 gene products are related to efficient viral DNA replication.

Cell Line↗
PubMed1997

Xenopus Pax-2 displays multiple splice forms during embryogenesis and pronephric kidney development.

Kidney organogenesis is initiated with the formation of the pronephric kidney and requires Pax-2 gene function. We report here the cloning and characterization of Pax-2 cDNAs from the frog Xenopus laevis, a model system suitable for the study of early kidney organogenesis. We show that expression of Xenopus Pax-2 (XPax-2) genes was confined to the nervous system, sensory organs, the visceral arches, and the developing excretory system. DNA sequencing of XPax-2 cDNAs isolated from head and pronephric kidney libraries revealed seven novel alternatively spliced Pax-2 isoforms. They all retain DNA-binding domains, but can differ significantly in their C termini with some isoforms containing a novel Pax-2 exon. We investigated the spectrum of XPax-2 splice events in pronephric kidneys, animal cap cultures and in whole embryos. Splicing of XPax-2 transcripts was found to be extensive and temporally regulated during Xenopus embryogenesis. Since all investigated tissues expressed essentially the full spectrum of XPax-2 splice variants, we conclude that splicing of XPax-2 transcripts does not occur in a tissue-specific manner.

Alternative Splicing↗
PubMed1987

Evolutionary conservation of the insulinoma gene rig and its possible function.

We have identified a gene, rig (rat insulinoma gene), that is activated in chemically induced rat insulinomas but not in normal pancreatic islets or in regenerating islets. In the present study, we have found that the insulinoma gene was activated in a BK virus-induced hamster insulinoma cell line and in a spontaneously occurring human insulinoma. From the hamster and human insulinoma cDNA libraries, rig homologues were isolated, and their nucleotide sequences were determined. In the same manner as the rat gene, both hamster and human homologues contained one open reading frame of 435 nucleotides, differing by 32- and 41-base substitutions, respectively. All the base substitutions were same-sense mutations. Accordingly, the deduced 145-amino acid sequence remained invariant in hamster, human, and rat insulinomas, suggesting that rig has evolved under extraordinarily strong selective constraints. Computerized structure analysis indicated that rig-encoded protein is a possible DNA-binding protein. The antisense oligodeoxyribonucleotide complementary to hamster rig mRNA was synthesized and injected into the hamster insulinoma cells. The antisense rig oligodeoxyribonucleotide inhibited DNA synthesis in the insulinoma cells, whereas the sense rig oligodeoxyribonucleotide or antisense insulin oligodeoxyribonucleotide had no inhibitory effect. These results strongly suggest that the activation of rig is both common and potentially significant in the oncogenic growth of pancreatic B cells of islets of Langerhans.

Adenoma, Islet Cell↗
PubMed1998

LUSH odorant-binding protein mediates chemosensory responses to alcohols in Drosophila melanogaster.

The molecular mechanisms mediating chemosensory discrimination in insects are unknown. Using the enhancer trapping approach, we identified a new Drosophila mutant, lush, with odorant-specific defects in olfactory behavior. lush mutant flies are abnormally attracted to high concentrations of ethanol, propanol, and butanol but have normal chemosensory responses to other odorants. We show that wild-type flies have an active olfactory avoidance mechanism to prevent attraction to concentrated alcohol, and this response is defective in lush mutants. This suggests that the defective olfactory behavior associated with the lush mutation may result from a specific defect in chemoavoidance. lush mutants have a 3-kb deletion that produces a null allele of a new member of the invertebrate odorant-binding protein family, LUSH. LUSH is normally expressed exclusively in a subset of trichoid chemosensory sensilla located on the ventral-lateral surface of the third antennal segment. LUSH is secreted from nonneuronal support cells into the sensillum lymph that bathes the olfactory neurons within these sensilla. Reintroduction of a cloned wild-type copy of lush into the mutant background completely restores wild-type olfactory behavior, demonstrating that this odorant-binding protein is required in a subset of sensilla for normal chemosensory behavior to a subset of odorants. These findings provide direct evidence that odorant-binding proteins are required for normal chemosensory behavior in Drosophila and may partially determine the chemical specificity of olfactory neurons in vivo.

Alcohols↗
PubMed1996

RAD9 and DNA polymerase epsilon form parallel sensory branches for transducing the DNA damage checkpoint signal in Saccharomyces cerevisiae.

In response to DNA damage and replication blocks, yeast cells arrest at distinct points in the cell cycle and induce the transcription of genes whose products facilitate DNA repair. Examination of the inducibility of RNR3 in response to UV damage has revealed that the various checkpoint genes can be arranged in a pathway consistent with their requirement to arrest cells at different stages of the cell cycle. While RAD9, RAD24, and MEC3 are required to activate the DNA damage checkpoint when cells are in G1 or G2, POL2 is required to sense UV damage and replication blocks when cells are in S phase. The phosphorylation of the essential central transducer, Rad53p, is dependent on POL2 and RAD9 in response to UV damage, indicating that RAD53 functions downstream of both these genes. Mutants defective for both pathways are severely deficient in Rad53p phosphorylation and RNR3 induction and are significantly more sensitive to DNA damage and replication blocks than single mutants alone. These results show that POL2 and RAD9 function in parallel branches for sensing and transducing the UV DNA damage signal. Each of these pathways subsequently activates the central transducers Mec1p/Esr1p/Sad3p and Rad53p/Mec2p/Sad1p, which are required for both cell-cycle arrest and transcriptional responses.

Cell Cycle↗