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Expression of immune responsive genes in cell lines from two different Anopheline species.

Malaria infection results in increased expression of immune responsive genes, including those encoding antimicrobial peptides such as Gambicin (Gam1) and Cecropin A (Cec1). Understanding how these genes are regulated will provide insights how the mosquito immune system is activated by Plasmodium. We previously have shown that Cec1 was primarily regulated by the Imd-Relish (REL2) pathway in the Anopheles gambiae Sua1B cell line. We show here that expression of Defensin A (Def1) and Gam1 was reduced after RNA interference against components of the Imd-REL2 pathway in An. gambiae cell lines. Interestingly, promoter reporters of these antimicrobial peptides were expressed at very low level in the cell line MSQ43 from Anopheles stephensi. Surprisingly, over-expression of either NF-kappaB transcription factor REL1 or REL2 alone is sufficient to induce the expression of Cec1, Gam1 and Def1. These results suggest that expression of these antimicrobial peptides (AMP) in vivo may be regulated by both the Toll and Imd pathways. We also show here for the first time that Tep4, a gene encoding a thioester containing protein, is regulated by REL2. Taken together, these results suggest that there are significant overlaps of genes regulated by the Toll-Rel1 and Imd-Rel2 pathways. Further, the different expression patterns in two different Anopheline cell lines provide a platform to identify other key positive and negative regulators of the antimicrobial peptide genes.

Animals↗

Molecular characterization and placental expression of HERV-W, a new human endogenous retrovirus family.

The multiple sclerosis-associated retrovirus (MSRV) isolated from plasma of MS patients was found to be phylogenetically and experimentally related to human endogenous retroviruses (HERVs). To characterize the MSRV-related HERV family and to test the hypothesis of a replication-competent HERV, we have investigated the expression of MSRV-related sequences in healthy tissues. The expression of MSRV-related transcripts restricted to the placenta led to the isolation of overlapping cDNA clones from a cDNA library. These cDNAs spanned a 7.6-kb region containing gag, pol, and env genes; RU5 and U3R flanking sequences; a polypurine tract; and a primer binding site (PBS). As this PBS showed similarity to avian retrovirus PBSs used by tRNATrp, this new HERV family was named HERV-W. Several genomic elements were identified, one of them containing a complete HERV-W unit, spanning all cDNA clones. Elements of this multicopy family were not replication competent, as gag and pol open reading frames (ORFs) were interrupted by frameshifts and stop codons. A complete ORF putatively coding for an envelope protein was found both on the HERV-W DNA prototype and within an RU5-env-U3R polyadenylated cDNA clone. Placental expression of 8-, 3.1-, and 1.3-kb transcripts was observed, and a putative splicing strategy was described. The apparently tissue-restricted HERV-W long terminal repeat expression is discussed with respect to physiological and pathological contexts.

Amino Acid Sequence↗

Genotypic and phenotypic comparisons of de novo and acquired melphalan resistance in an isogenic multiple myeloma cell line model.

Cancer cell adhesion confers a transient, de novo drug-resistant phenotype referred to as cell adhesion-mediated drug resistance (CAM-DR). In this report, we extend the CAM-DR phenotype to primary specimens from patients with myeloma, providing further evidence that CAM-DR is a viable clinical form of drug resistance. To examine mechanisms of cellular resistance to melphalan, we compared genotypic and phenotypic profiles of acquired and de novo melphalan resistance in an isogenic human myeloma cell line. Acquired melphalan resistance (8226/LR5) was associated with decreased drug-induced DNA damage and a complex gene expression profile showing that genes involved in the Fanconi anemia DNA repair pathway are increased in the LR5 cells compared with drug-sensitive or adherent cells. In contrast, cells adhered to fibronectin accumulate similar amounts of DNA damage compared with drug-sensitive cells but are protected from melphalan-induced mitochondrial perturbations and caspase activation. Levels of the proapoptotic protein Bim were significantly reduced in adherent cells. Gene expression changes associated with de novo resistance were significantly less complex compared with acquired resistance, but a significant overlap in gene expression was noted involving cholesterol synthesis. We propose that myeloma cell adhesion promotes a form of de novo drug resistance by protecting cells from melphalan-induced cytotoxic damage and that this transient protection allows cells to acquire a more permanent and complex drug resistance phenotype associated with a reduction in drug induced DNA damage.

Antineoplastic Agents, Alkylating↗

Porcine relaxin. Gene structure and expression.

A porcine genomic cosmid library was constructed to study the structure and regulation of the porcine relaxin gene. Two overlapping cosmids containing relaxin-specific sequences were isolated, and a 9-kilobase BamHI fragment containing the porcine relaxin gene was sequenced. The sequence derived from these cosmids was used to characterize the relaxin gene transcription unit utilized in the pregnant ovary. Analysis of chromosomal and cosmid DNA has shown that porcine relaxin is encoded by a single copy gene, comprising two exons separated by a 5.5-kilobase intron. The gene contains elements representing two classes of repeated sequences homologous with the human Alu and Kpn families. The transcriptional initiation site of this gene was located by primed extension and direct sequencing of relaxin-specific cDNA. Furthermore, the promoter elements were mapped and sequenced, and transcriptional activity was demonstrated. The promoter contained sequences with homology to regions conserved within retroviral long terminal repeats and within the adenovirus E4 promoter.

Animals↗

Association of a human H1 histone gene with an H2A pseudogene and genes encoding H2B.1 and H3.1 histones.

A cluster of human histone genes was found on three overlapping clones isolated from cosmid and bacteriophage libraries. These three overlapping segments of the human genome comprise genes coding for H3.1, an H2A pseudogene, and an H2B.1 gene downstream of the previously characterized H1.2 gene. The cosmid clone covers 30 kb upstream of the H1.2 gene and overlaps with two phage clones covering the core histone genes and the pseudogene. The same arrangement of an H3 gene, an H2A pseudogene and an H2B gene downstream of an H1 gene has been described within a mouse histone gene cluster [Yang et al.:J Biol Chem 262:17118-17125, 1987; Gruber et al.:Gene 95:303-304, 1990].

Amino Acid Sequence↗

Functional genomic analysis of the AUXIN RESPONSE FACTOR gene family members in Arabidopsis thaliana: unique and overlapping functions of ARF7 and ARF19.

The AUXIN RESPONSE FACTOR (ARF) gene family products, together with the AUXIN/INDOLE-3-ACETIC ACID proteins, regulate auxin-mediated transcriptional activation/repression. The biological function(s) of most ARFs is poorly understood. Here, we report the identification and characterization of T-DNA insertion lines for 18 of the 23 ARF gene family members in Arabidopsis thaliana. Most of the lines fail to show an obvious growth phenotype except of the previously identified arf2/hss, arf3/ett, arf5/mp, and arf7/nph4 mutants, suggesting that there are functional redundancies among the ARF proteins. Subsequently, we generated double mutants. arf7 arf19 has a strong auxin-related phenotype not observed in the arf7 and arf19 single mutants, including severely impaired lateral root formation and abnormal gravitropism in both hypocotyl and root. Global gene expression analysis revealed that auxin-induced gene expression is severely impaired in the arf7 single and arf7 arf19 double mutants. For example, the expression of several genes, such as those encoding members of LATERAL ORGAN BOUNDARIES domain proteins and AUXIN-REGULATED GENE INVOLVED IN ORGAN SIZE, are disrupted in the double mutant. The data suggest that the ARF7 and ARF19 proteins play essential roles in auxin-mediated plant development by regulating both unique and partially overlapping sets of target genes. These observations provide molecular insight into the unique and overlapping functions of ARF gene family members in Arabidopsis.

Arabidopsis↗

Polyadenylation creates the discriminator nucleotide of chicken mitochondrial tRNA(Tyr).

In the chicken mitochondrial genome, the gene for tRNA(Tyr) overlaps by one nucleotide with the downstream tRNA(Cys) gene, which is located on the same strand. The overlapping nucleotide, a guanosine residue, thus encodes both the discriminator base of the tRNA(Tyr) and the 5'base of the tRNA(Cys). When cDNA clones of circularized forms of the tRNA(Tyr) are analyzed, the discriminator nucleotide is an adenosine residue rather than the genomically encoded guanosine. Thus, the tRNA(Tyr) is subjected to an RNA editing activity similar to that shown to exist in the mitochondria of two other animal species. Interestingly, some cDNA clones have several adenosine residues at their 3'-ends instead of the expected CCA-sequence. Furthermore, a review of sequence data from animal mitochondrial genomes suggests that only tRNAs whose discriminator bases are adenosines tend to have genes that overlap with downstream genes. Thus, polyadenylation seems to be a major component of the RNA editing machinery that affects overlapping genes in animal mitochondria.

Animals↗

Genetic recombination in Bacillus subtilis 168: effect of recN, recF, recH and addAB mutations on DNA repair and recombination.

A recN- (recN1) strain of Bacillus subtilis was constructed. The effects of this and recF, recH and addAB mutations on recombination proficiency were tested. Mutations in the recN, recF, recH and addAB genes, when present in an otherwise Rec+ B. subtilis strain, did not affect genetic exchange. Strains carrying different combinations of mutations in these genes were constructed and examined for their sensitivity to 4-nitroquinoline-1-oxide (4NQO) and recombination proficiency. The recH mutation did not affect the 4NQO sensitivity of recN and recF cells and it only marginally affected that of addA addB cells. However, it reduced genetic recombination in these cells 10(2)- to 10(4)-fold. The addA addB mutations increased the 4NQO sensitivity of recF and recN cells, but completely blocked genetic recombination of recF cells and marginally affected recombination in recN cells. The recN mutation did not affect the recombinational capacity of recF cells. These data indicate that the recN gene product is required for DNA repair and recombination and that the recF, recH and addAB genes provide overlapping activities that compensate for the effects of single mutants proficiency. We proposed that the recF, recH, recB and addA gene products define four different epistatic groups.

Bacillus subtilis↗

Gene expression profiling in human peripheral blood mononuclear cells using high-density filter-based cDNA microarrays.

Microarray technology has provided the ability to analyse the expression profiles for thousands of genes in parallel. The need for highly specialised equipment to use certain types of microarrays has restricted the application of this technology to a small number of dedicated laboratories. High-density filter-based cDNA microarrays provide a low-cost option for performing high-throughput gene expression analysis. We have used a model system in which filter-based cDNA microarrays representing over 4000 known human genes were used to monitor the kinetics of gene expression in human peripheral blood mononuclear cells (PBMCs) stimulated with phytohaemagluttinin (PHA). Using software-based cluster analysis, we identified 104 genes that altered in expression levels in response to PHA stimulation of PBMCs and showed that there was a considerable overlap between genes with similar temporal expression profiles and similar functional roles. Comparison of microarray quantitation with quantitative PCR showed almost identical expression profiles for a number of genes. Coupled with the fact that our findings are in agreement with a large number of independent observations, we conclude that the use of filter-based cDNA microarrays is a valid and accurate method for high-throughput gene expression profiling.

Cells, Cultured↗

Collier, a novel regulator of Drosophila head development, is expressed in a single mitotic domain.

BACKGROUND: Segmentation of the Drosophila embryo is based on a cascade of hierarchical gene interactions that is initiated by maternal morphogens; these interactions define spatially restricted domains of zygotic gene expression within the blastoderm. Although the hierarchy of the segmentation genes that subdivide the trunk is well established, the patterning of the head is less well understood. Seven head segments can be assigned on the basis of metameric patterns of segment-polarity gene expression and internal sensory organs. The domains of expression of head gap-like genes broadly overlap, with their posterior margins out of phase by one segment. Taken together with the lack of pair-rule gene expression in the head, these observations led to the suggestion that head gap genes act in a combinatorial manner, determining head segmental borders and segmental identity at the same time. RESULTS: We have identified a new Drosophila gene, collier (col), whose expression at the blastoderm stage is restricted to a single stripe of cells corresponding to part of the intercalary and mandibular segment primordia, possibly parasegment O. Reduction of col activity in early gastrula embryos by antisense RNA expression results in a specific lack of head structures derived from these segments. The expression of col coincides with a mitotic domain, which supports the proposal that cells in this domain undergo a concerted mitotic and differentiation program that is orchestrated at the transcriptional level. Col is an ortholog of mammalian early B-cell factor/Olfactory-1. These proteins define a new family of transcription factors that contain a helix-loop-helix dimerization motif and a new type of DNA-binding domain that is highly conserved during evolution. CONCLUSIONS: Here we describe Col, the first Drosophila member of a new family of transcription factors. Col may act as a "second-level regulator' of head patterning. The structural conservation of Col during evolution raises the questions of its conservation of function in head specification and its interactions with other factors conserved between insects and vertebrates.

Amino Acid Sequence↗

Genotype/phenotype and penetrance studies in melanoma families with germline CDKN2A mutations.

Patients with a family history of melanoma are at increased risk of this tumor. Those family members who also have the atypical mole syndrome are commonly targeted for screening in the belief that they are more likely to be mutant gene carriers. We have correlated the atypical mole syndrome phenotype and gene carrier status in five families with germline CDKN2A mutations and shown that family members with the atypical mole syndrome were three times more likely to be mutant gene carriers than their relatives who did not have the atypical mole syndrome (odds ratio 3.4; confidence interval 1.0-11. 1), supporting the view that CDKN2A is nevogenic. Individual characteristics which best predicted mutant gene carrier status were: nevi on the buttocks (odds ratio 4.4; confidence interval 1. 6-12.4), nevi on the feet (odds ratio 4.2; confidence interval 1. 4-12.5), total nevus number being at least 100 (nevi > or = 2 mm in diameter) (odds ratio 3.4; confidence interval 1.0-11.1) and two or more clinically atypical nevi (odds ratio 3.1; confidence interval 1. 1-9.0). Gene carriers were also significantly more likely to have noticeable freckling and possibly also Fitzpatrick skin types 1-3. The overlap between gene carriers and nongene carriers was, however, marked: the atypical mole syndrome did not clearly differentiate mutant gene carriers from those with a normal gene. This study is of significance to clinicians as the clinical practice of using the atypical mole syndrome to identify particular family members for surveillance is shown to be inappropriate. Until formal gene testing is available, all members of families with an excessive number of melanoma cases should be treated as potential mutation carriers at increased risk of melanoma.

Genes, p16↗

Isolation and characterization of genomic clones covering the chicken vitellogenin gene.

A series of overlapping recombinant clones, which cover the vitellogenin gene, has been isolated from a phage-lambda linked chicken gene library. The DNA of the overlapping clones spans 28 kb of contiguous DNA sequences in the chicken genome. Electron microscopic analysis of hybrids between vitellogenin mRNA and the genomic clones indicates that the chicken vitellogenin gene has a length of approximately 22 kb, about 3.8 times the size of the mRNA. The mRNA sequence is interrupted by at least 33 intervening sequences (introns). Comparison with the vitellogenin gene A2 from Xenopus laevis (Wahli et al., 1980, Cell 20: 107-117) indicates conservation of the number and length of the exons during evolution. Heteroduplex analysis reveals a short stretch of sequence homology between the genes from chicken and frog.

Animals↗

Mobilization of the non-conjugative plasmid RSF1010: a genetic and DNA sequence analysis of the mobilization region.

The entire region required for mobilization of the non-conjugative plasmid RSF1010 has been cloned into a mobilization-deficient pBR322 derivative. The segment of DNA cloned was approximately 1.8 kb and included the origin of conjugal DNA transfer (oriT). The DNA sequence of the mobilization region has been determined, and revealed the presence of several overlapping reading frames. The isolation and mapping of both Tn1725 and BamH1-linker insertions and comparison with the DNA sequence data has allowed the identification of three genes required for mobilization. Two of these genes are overlapping and encode proteins of 16 kDa and greater than 65 kDa (although the truncated protein is functional, the gene extends outside the region cloned). The third gene is transcribed in the opposite direction. Promoters capable of transcribing these genes were located by S1 mapping in the inter-cistronic region between these divergently transcribed genes. The oriT site is located in this region, and the transcriptional patterns observed for mob+ and mob- plasmids implied that the promoters may be regulated by two of the mobilization proteins binding to the oriT site.

Base Sequence↗

The HOX genes are expressed, in vivo, in human tooth germs: in vitro cAMP exposure of dental pulp cells results in parallel HOX network activation and neuronal differentiation.

Homeobox-containing genes play a crucial role in odontogenesis. After the detection of Dlx and Msx genes in overlapping domains along maxillary and mandibular processes, a homeobox odontogenic code has been proposed to explain the interaction between different homeobox genes during dental lamina patterning. No role has so far been assigned to the Hox gene network in the homeobox odontogenic code due to studies on specific Hox genes and evolutionary considerations. Despite its involvement in early patterning during embryonal development, the HOX gene network, the most repeat-poor regions of the human genome, controls the phenotype identity of adult eukaryotic cells. Here, according to our results, the HOX gene network appears to be active in human tooth germs between 18 and 24 weeks of development. The immunohistochemical localization of specific HOX proteins mostly concerns the epithelial tooth germ compartment. Furthermore, only a few genes of the network are active in embryonal retromolar tissues, as well as in ectomesenchymal dental pulp cells (DPC) grown in vitro from adult human molar. Exposure of DPCs to cAMP induces the expression of from three to nine total HOX genes of the network in parallel with phenotype modifications with traits of neuronal differentiation. Our observations suggest that: (i) by combining its component genes, the HOX gene network determines the phenotype identity of epithelial and ectomesenchymal cells interacting in the generation of human tooth germ; (ii) cAMP treatment activates the HOX network and induces, in parallel, a neuronal-like phenotype in human primary ectomesenchymal dental pulp cells.

Adolescent↗

Identification of target genes of the p16INK4A-pRB-E2F pathway.

Deregulation of the retinoblastoma protein (pRB) pathway is a hallmark of human cancer. The core members of this pathway include the tumor suppressor protein, pRB, which through binding to a number of cellular proteins, most notably members of the E2F transcription factor family, regulates progression through the cell division cycle. With the aim of identifying transcriptional changes provoked by deregulation of the pRB pathway, we have used cell lines that conditionally express a constitutively active phosphorylation site mutant of pRB (pRBDeltaCDK) or p16INK4A (p16). The expression of pRBDeltaCDK and p16 resulted in significant repression and activation of a large number of genes as measured by high density oligonucleotide array analysis. Transcriptional changes were found in genes that are essential for DNA replication and cell proliferation. In agreement with previous results, we found a high degree of overlap between genes regulated by p16 and pRB. Data we have obtained previously for E2F family members showed that 74 of the genes repressed by pRB and p16 were induced by the E2Fs and 23 genes that were induced by pRB and p16 were repressed by the E2Fs. Thus, we have identified 97 genes as physiological targets of the pRB pathway, and the further characterization of these genes should provide insights into how this pathway controls proliferation. We show that Gibbs sampling detects enrichment of several sequence motifs, including E2F consensus binding sites, in the upstream regions of these genes and use this enrichment in an in silico filtering process to refine microarray derived gene lists.

Amino Acid Motifs↗

Coexpression of liver-specific and growth-induced genes in perinatal and regenerating liver: attainment and maintenance of the differentiated state during rapid proliferation.

The liver shows maximal cellular growth during fetal development and after partial hepatectomy. Exploring overlaps in gene expression patterns in these two types of hepatic growth may provide insight into common regulatory pathways. The expression of a large number of growth-induced and liver-specific genes induced in liver regeneration has been examined in the perinatal liver from several days prenatal to 4 weeks postnatal when the major growth phase of the liver ceases. As in liver regeneration, many growth-induced genes, such as PRL-1 and beta-actin, are expressed at a high level throughout the temporal course of liver development and correlate with the proliferative state. The level of fetal liver expression of these genes is similar to peak expression found in the regenerating liver, suggesting that common pathways of transcriptional regulation exist in the two types of proliferation. A subset of liver-restricted immediate-early genes including, IGFBP-1, CL-6, and glucose-6-phosphatase (G6Pase) are induced in regenerating liver and may be important in maintaining hepatic metabolism during regeneration. In developing liver, these genes are expressed primarily in the perinatal period but, unlike the regenerating liver, are not coinduced. For instance, at birth, G6Pase is induced, whereas CL-6 is downregulated. In situ analyses confirm that a proliferation associated gene PRL-1 is expressed in multiple cell types throughout the developing liver, whereas the expression of liver-specific genes is confined to hepatocytes. Taken together, these findings imply that significant similarities and differences in transcriptional regulation and hormonal milieu exist in liver during regeneration and development.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Interactive regulation of Azorhizobium nifA transcription via overlapping promoters.

The Azorhizobium nifA promoter (PnifA) is positively regulated by two physiological signal transduction pathways, NtrBC, which signals anabolic N status, and FixLJK, which signals prevailing O2 status. Yet, PnifA response (gene product per unit time) to these two activating signals together is more than twice that of the summed, individual signals. In the absence of NIFA, a negative PnifA autoregulator, the fully induced PnifA response is more than 10-fold greater than that of summed, individual signals. Given this synergism, these two signal transduction pathways must interactively regulate PnifA activity. PnifA carries three cis-acting elements, an anaerobox, which presumably binds FIXK, a NIFAbox, which presumably binds NIFA itself, and a sigma 54 box, which presumably binds sigma 54 initiator, a subunit of RNA polymerase. For combinatorial analysis, single, double, and triple promoter mutations were constructed in these cis-acting elements, and PnifA activities were measured in six different trans-acting background, i.e., fixK, fixJ, nifA, ntrC, rpoF, and wild type. Under all physiological conditions studied, high-level PnifA activity required both FIXK in trans and the anaerobox element in cis. Surprisingly, because PnifA was hyperactive with a mutated sigma 54box, this cis-acting element mediates both negative and positive control. Because PnifA hyperactivity also required a wild-type upstream NIFAbox element, even in the absence of NIFA, a second upstream nifA transcription start superimposed on the NIFAbox element was hypothesized. When nifA mRNA 5' start points were mapped by primer extension, both a minor upstream transcript(s) starting 45 bp distal to the anaerobox and a major downstream transcript starting 10 bp distal to the sigma 54 box were observed. In Azorhizobium, RNA polymerase sigma 54 initiator subunits are encoded by a multigene family, which includes rpoF and rpoN genes. Because rpoF mutants show an Ntr+ phenotype, whereas rpoN mutants are Ntr-, multiple sigma 54 initiators are functionally distinct. Two independent rpoF mutants both show a tight Nif- phenotype. Moreover, rpoF product sigma 54F is absolutely required for high-level PnifA activity. In summary, the Azorhizobium nifA gene carries overlapping housekeeping-type and sigma 54-type promoters which interactively respond to different signals. Effectively, the upstream, housekeeping-type promoter responds to FIXK and positively regulates the downstream, sigma 54-type promoter. The downstream, sigma 54-type promoter responds to NTRC and negatively regulates the upstream, housekeeping-type promoter. In terms of transcript yield, the upstream, housekeeping-type promoter is therefore weak, and the downstream, sigma 54-type promoter is strong.

Bacterial Proteins↗

Overlap and cotranscription of the genes for the beta and epsilon subunits of tobacco chloroplast ATPase.

The nucleotide sequences of the genes for the beta and epsilon subunits of tobacco chloroplast ATPase have been determined. The coding regions for the beta and epsilon subunits contain 1494 bp (498 codons) and 399 bp (133 codons), respectively. The 3' end of the beta-coding region overlaps by one nucleotide with the 5' end of the epsilon-coding region. The overlapping termination and initiation codons are ATGA. The beta and epsilon genes are cotranscribed as a 2.7-kb polycistronic mRNA. The amount of the beta and epsilon mRNA in the chloroplast is about one-twentieth that of the LS mRNA.

Base Sequence↗