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Reconstructing dynamic regulatory maps.

Even simple organisms have the ability to respond to internal and external stimuli. This response is carried out by a dynamic network of protein-DNA interactions that allows the specific regulation of genes needed for the response. We have developed a novel computational method that uses an input-output hidden Markov model to model these regulatory networks while taking into account their dynamic nature. Our method works by identifying bifurcation points, places in the time series where the expression of a subset of genes diverges from the rest of the genes. These points are annotated with the transcription factors regulating these transitions resulting in a unified temporal map. Applying our method to study yeast response to stress, we derive dynamic models that are able to recover many of the known aspects of these responses. Predictions made by our method have been experimentally validated leading to new roles for Ino4 and Gcn4 in controlling yeast response to stress. The temporal cascade of factors reveals common pathways and highlights differences between master and secondary factors in the utilization of network motifs and in condition-specific regulation.

Algorithms↗

Divergent regulation of cytochrome P450 enzymes by morphine and pethidine: a neuroendocrine mechanism?

We have studied the effects of opiates on cytochrome P450 (CYP) expression in rats and, intriguingly, found that these compounds have divergent effects on this system. Morphine induced a selective down-regulation of hepatic 16 alpha-hydroxylation of androstenedione, which was associated with a reduction in the expression of the 16 alpha-hydroxylation-associated CYP2C11. In addition, other enzymes from the CYP2C, CYP3A, and CYP4A gene families were also suppressed, whereas CYP1A2, CYP2B1, and CYP2E1 were induced. These changes were reflected in the mRNA levels, indicating that they were due to alterations at the transcriptional level. alpha 2-Adrenoceptor blockade with yohimbine augmented the effects of morphine on proteins identified by antibodies against CYP2C11 and CYP3A, indicating an involvement or influence of the alpha 2-adrenergic receptor system. In contrast, pethidine had completely different effects. No CYPs were suppressed by this compound. CYP1A2, CYP2B1, CYP2C6, CYP2C7, CYP3A, and CYP4A1 were all induced by pethidine treatment, whereas CYP2C11 and CYP2E1 were unchanged. Our results demonstrate for the first time the divergent effects of pethidine and morphine on the hepatic CYP system, indicating that an opioid receptor mechanism is not involved. These differences are probably mediated by different influences on growth hormone secretion. The mechanisms by which this may occur are discussed.

Adrenal Glands↗

Chloroplast-expressed glutamine synthetase (ncpGS): potential utility for phylogenetic studies with an example from Oxalis (Oxalidaceae).

Chloroplast-expressed glutamine synthetase (ncpGS), a nuclear-encoded gene containing several introns, is introduced as a tool for phylogenetic studies at lower taxonomic levels. This gene is a member of a multigene family, but it diverged long ago from the cytosolic-expressed members of the family and appears to be single copy in the majority of taxa examined to date. The conservation of both coding sequence and position of introns has allowed the design of primers for use in a broad range of dicot taxa to amplify and sequence a region of ncpGS that contains four introns. The utility of this region in phylogenetic studies of congeneric species is illustrated by an example using eight Oxalis species. The four introns in these taxa are typical in size (76 to 136 bp), base composition (high T content), and structure (e.g., sequence of splice sites and putative branch points) for plant internal introns. Levels of variation among these ncpGS sequences compare favorably with those of the internal transcribed spacer of nuclear ribosomal DNA (ITS) from the same taxa, and results of phylogenetic analysis of ncpGS data are generally congruent with previous results using ITS.

Base Sequence↗

Mammalian sperm proteins are rapidly evolving: evidence of positive selection in functionally diverse genes.

A growing number of genes involved in sex and reproduction have been demonstrated to be rapidly evolving. Here, we show that genes expressed solely in spermatozoa represent a highly diverged subset among mouse and human tissue-specific orthologs. The average rate of nonsynonymous substitutions per site (K(a)) is significantly higher in sperm proteins (mean K(a) = 0.18; N = 35) than in proteins expressed specifically in all other tissues (mean K(a) = 0.074; N = 473). No differences, however, are found in the synonymous substitution rate (K(s)) between tissues, suggesting that selective forces, and not mutation rate, explain the high rate of replacement substitutions in sperm proteins. Four out of 19 sperm-specific genes with characterized function demonstrated evidence of strong positive Darwinian selection, including a protein involved in gene regulation, Protamine-1 (PRM1), a protein involved in glycolysis, GAPDS, and two egg-binding proteins, Adam-2 precursor (ADAM2) and sperm-adhesion molecule-1 (SAM1). These results demonstrate the rapid evolution of sperm-specific genes and highlight the molecular action of sexual selection on a variety of characters involved in mammalian sperm function.

Amino Acid Substitution↗

p53 expression in cultured blood human monocytes infected with mycobacterial strains.

BACKGROUND: An upregulation of the cell-cycle associated proteins p53 and p21/Waf1/Cip1 induced by mycobacteria was previously reported. We aimed to evaluate the expression of such proteins in peripheral blood human monocyte cultures infected with strains of different mycobacterial pathogens. METHODS: The study relied on the immunocytochemical determination of p53, p21/Waf1/Cipl, bcl-2 and on the Tunel detection of apoptosis in monocytes populations cultured on four-welled chamber slides (10(6) cells/well) infected with Mycobacterium tuberculosis, M. bovis and M. avium for four consecutive days (mycobacterium/monocyte ratio 10:1). The results were expressed as mean values and SD of the percentages of stainings recorded in five fields per slide. RESULTS: The statistical analysis with Fischer test demostrated that at most sampling times the p53 and p21/Waf1/Cip1 expression and the apoptosis index were significantly higher in M. tuberculosis infected cultures than in controls (p<0.05). The M. bovis related picture diverged from the previous one for a lower p53 expression (p<0.05) at all sampling times. The M. avium infected culture values did not diverge significantly from the controls. CONCLUSIONS: The p53 and p21/Wafl/Cipl upregulation is compatible with both host defense strategies and pathogen strategies (safeguard of intracellular sanctuaries). The discrepancies among different cultures suggest a direct relationship between p53 activation and mycobacterial ability to enter host cells.

Apoptosis↗

Construction of a bidirectional promoter probe vector and its use in analysing nod gene expression in Rhizobium loti.

A broad-host-range bidirectional promoter reporter vector, pSPV4, has been constructed to analyse the activity of cloned divergent regulatory regions. Plasmid pSPV4 contains a pair of divergent promoterless reporter genes (lacZ and gusA) that are bisected by an extensive multiple cloning site. [corrected] The transcriptional fusion vector pSPV4 has a distinct advantage over unidirectional promoter probe vectors in that it can determine the activity of a cloned bidirectional regulatory region simultaneously in both directions using the same set of cells. The relative activity of each of the divergent promoters, and hence the reporter genes, is therefore not a function of the particular growth state of the culture. To demonstrate the utility of pSPV4, the promoter activity of two Rhizobium loti nod regulatory regions were examined. Although the nod gene organisation in this species is unusual, the promoter activity of these two divergent nod regulatory regions is consistent with the conventional mode of nod expression (constitutive towards the regulatory nodD gene and inducible in the divergent nod gene direction). The construction of the bidirectional reporter vector, pSPV4, involved two intermediary plasmids, pSPV1 and pSPV2. Both of these constructs will also be useful for other researchers, especially given the trend towards the utilisation of gusA as a reporter gene.

Acyltransferases↗

px(2), the newly identified gene in Rhizobium leguminosarum, is characterized to enhance its adjacent nodF expression.

nodFEL operon is one of the NodD-dependent inducible nod operons that is clustered on the symbiosis plasmid of Rhizobium leguminosarum biovar viciae. A recent study on the upstream regulatory region of nodFEL operon had identified a new promoter, which was responsible for the transcription of a 0.72 Kb RNA molecule, called px(2), in the opposite direction to nodF. This new promoter was further characterized to overlap with that of nodF and its in vitro transcription was inhibited by another newly identified nod regulator, Px. In this paper we report that the sequence analysis of the px(2) transcript revealed only one open reading frame (ORF(66)), corresponding to a polypeptide of 66 amino acids. Moreover, the increase in px(2) copy numbers enhanced the in vivo inducible expression level of nodF, whereas the frame-shift mutation of ORF(66) eliminated such effect, providing evidence that px(2) is responsible for specifically upregulating nodF expression. This result also raises the big possibility that px(2) encodes this polypeptide. A model for coordinated expression of px(2) and nodF, transcribed divergently from each other, is proposed.

Amino Acid Sequence↗

The ascidian genome contains another T-domain gene that is expressed in differentiating muscle and the tip of the tail of the embryo.

The vertebrate Brachyury (T) gene is transiently expressed in nascent and migrating mesoderm, in the differentiating notochord, and in the tail bud, reflecting its independent functions. In contrast, the expression of an ascidian Brachyury gene (As-T) is restricted to differentiating notochord. The present study revealed that the genome of Halocynthia roretzi contains another T-domain gene (As-T2) which encodes a divergent T-domain protein. The transient expression of As-T2 was detected in the endoderm- and muscle-lineage blastomeres of the early embryo and the transcript was retained by involuting and differentiating muscle cells until it became undetectable by the mid-tailbud stage. In addition, As-T2 was expressed transiently in cells that form the tip of the newly forming tail. Interestingly, the combined pattern of spatial expression of As-T and As-T2 appears to correspond to that of a single vertebrate Brachyury gene.

Amino Acid Sequence↗

Expression of two csg operons is required for production of fibronectin- and congo red-binding curli polymers in Escherichia coli K-12.

Two divergently transcribed operons in Escherichia coli required for the expression of fibronectin- and Congo red-binding curli polymers were identified and characterized by transposon mutagenesis, sequencing and transcriptional analyses, as well as for their ability to produce the curli subunit protein. The csgBA operon encodes CsgA, the major subunit protein of the fibre, and CsgB, a protein with sequence homology to CsgA. A non-polar csgB mutant is unaffected in its production of CsgA, but the subunit protein is not assembled into insoluble fibre polymers. A third open reading frame, orfC, positioned downstream of csgA may affect some functional property of curli since an insertion in this putative gene abolishes the autoagglutinating ability typical of curliated cells without affecting the production of the fibre. The promoter for the oppositely transcribed csgDEFG operon was identified by primer extension and shown, like the csgBA promoter, to be dependent upon the alternate stationary phase-specific sigma factor sigma s in wild-type cells, but not in mutants lacking the nucleoid associated protein H-NS. Insertions in csgD abolish completely trancription from the csgBA promoter. Therefore, any regulatory effect on the csgBA promoter might be secondary to events controlling the csgDEFG promoter and/or activation of CsgD. Insertions in csgE, csgF and csgG abolish curli formation but allow CsgA expression suggesting that one or more of these gene products are involved in secretion/assembly of the CsgA subunit protein. No amino acid sequence homologies were found between the CsgE, CsgF and CsgG proteins and secretion/assembly proteins for other known bacterial fibres, suggesting that the formation of curli follows a novel pathway.

Bacterial Proteins↗

A cyclic AMP receptor protein mutant that constitutively activates an Escherichia coli promoter disrupted by an IS5 insertion.

Previously an Escherichia coli mutant that had acquired the ability to grow on propanediol as the sole carbon and energy source was isolated. This phenotype is the result of the constitutive expression of the fucO gene (in the fucAO operon), which encodes one of the enzymes in the fucose metabolic pathway. The mutant was found to bear an IS5 insertion in the intergenic regulatory region between the divergently oriented fucAO and fucPIK operons. Though expression of the fucAO operon was constitutive, the fucPIK operon became noninducible such that the mutant could no longer grow on fucose. A fucose-positive revertant which was found to contain a suppressor mutation in the crp gene was selected. Here we identify this crp mutation, which results in a single amino acid substitution (K52N) that has been proposed previously to uncover a cryptic activating region in the cyclic AMP receptor protein (CRP). We show that the mutant CRP constitutively activates transcription from both the IS5-disrupted and the wild-type fucPIK promoters, and we identify the CRP-binding site that is required for this activity. Our results show that the fucPIK promoter, a complex promoter which ordinarily depends on both CRP and the fucose-specific regulator FucR for its activation, can be activated in the absence of FucR by a mutant CRP that uses three, rather than two, activating regions to contact RNA polymerase. For the IS5-disrupted promoter, which retains a single CRP-binding site, the additional activating region of the mutant CRP evidently compensates for the lack of upstream regulatory sequences.

Alleles↗

The T complex distorter 2 candidate gene, Dnahc8, encodes at least two testis-specific axonemal dynein heavy chains that differ extensively at their amino and carboxyl termini.

Homozygosity for the t haplotype allele of the testis-specifically expressed axonemal dynein heavy chain (axDHC) gene, Dnahc8, has been linked to male sterility resulting from aberrant sperm motility. However, the near absence of Dnahc8 expression has been associated with male sterility resulting from an early breakdown in sperm flagellar development. Although axDHCs are integral participants in flagellar motility, a role in flagellar morphogenesis has never been attributed to a member of this highly conserved gene family. To gain a better understanding of this presumed novel role for Dnahc8, we have studied the organization and expression of full-length Dnahc8(+) and Dnahc8(t) transcripts. Our results demonstrate the existence of at least two alternatively spliced, testis-specific Dnahc8 mRNAs transcribed from both the + and t alleles. A highly expressed isoform encodes a protein with significant homology nearly throughout to the gamma heavy chain of the Chlamydomonas axonemal outer arm dynein, while a more poorly expressed isoform codes for a protein whose sequence diverges significantly from that of other axDHCs at both its N and C termini. While in situ hybridization studies demonstrate that both mRNA species accumulate exclusively in mid to late spermatocytes, each isoform shows spatial independence. Additional experiments demonstrate the existence of a testis-expressed mRNA with no significant open reading frame, a portion of which is antisense to the 5'-untranslated region of the highly divergent Dnahc8 isoform. The cumulative data imply that Dnahc8 may have acquired functional plasticity in the testis through the tightly controlled expression of both typical and unusual isoforms.

Amino Acid Sequence↗

The somatic patient.

A significant proportion of patients seen in the Emergency Department will present with somatic complaints for which there is no apparent physiologic cause. Such patients may be divided into two broad categories: (1) those with symptoms and signs consciously synthesized by the patient, either for obvious secondary gain (malingering) or as a result of more subtle and complex motivations (factitious disorders); and (2) those patients with symptoms that are the unconscious expression of psychological stress (somatoform disorders). The somatoform disorders include (1) somatization disorder (characterized by a chronic history of numerous and widely divergent somatic complaints), (2) psychogenic pain disorder (somatization expressed in terms of persistent pain), (3) hypochondriasis (a conviction that one is diseased and disabled in conjunction with a well-focused constellation of supporting symptoms), and (4) conversion disorder (a single, usually nonpainful neurologic symptom, often with identifiable coping value for the patient). The first three disorders have been aggregately termed the "common somatization syndrome." Management of the somatically focused patient includes the communication of a caring attitude to the patient in conjunction with a cautious and diligent search for treatable medical or psychiatric illness. Resocialization and development of patient links with ongoing, nurturing nonmedical as well as medical support systems is of benefit.

Emergency Service, Hospital↗

IL-1 stimulates a diverging signaling pathway in EL4 6.1 thymoma cells. IL-2 release, but not IL-2 receptor expression, is sensitive to pertussis toxin.

We reassessed the involvement of Bordetella pertussis toxin (PTX)-sensitive proteins in the IL-1 signaling pathway on the responses induced by IL-1 on the murine thymoma cell line EL4 6.1. We demonstrate that the ADP-ribosyltransferase activity of PTX, and not its cell-anchoring B oligomer part, is responsible for the inhibition of IL-1-induced IL-2 release, since 1) the concentration of PTX (< or = 1 ng/ml) required to block the secretion is 100 to 1000 times lower than the concentration needed with the B oligomer; and 2) the mutated PT-9K/129G, devoid of ADP-ribosyltransferase activity, was inactive at 100 ng/ml. We found that partial ADP-ribosylation of the Gi2/Gi3 proteins before stimulation with IL-1 was sufficient to obtain full inhibition of IL-2 release. PTX did not however inhibit the appearance on the cell surface of the high affinity IL-2 receptors or the IL-2 release induced by PMA. In addition, we show that PTX prevented the expression of the IL-2 mRNA induced by IL-1, without affecting the binding of IL-2 specific nuclear factors to the T cell distal element of the IL-2 promoter. Furthermore, PTX also inhibited IL-1-induced proliferation of non-transformed thymocytes. In conclusion, our results demonstrate that IL-1-induced IL-2 release is sensitive to PTX-catalyzed ADP-ribosylation and that IL-1 activates a diverging pathway on EL4 6.1 cells.

Animals↗

Gene regulation and evolution in the chorion locus of Bombyx mori. Structural and developmental characterization of four eggshell genes and their flanking DNA regions.

We report on the detailed structural and developmental characterization of four chorion genes and a truncated pseudogene located within a 9.5 X 10(3) base chromosomal segment. These genes belong to the A and B multigene families and, like previously characterized moth chorion genes, are arranged in tightly linked pairs, which are divergently transcribed (A/B.L11 and A/B.L12). On the basis of their high degree of sequence divergence, the A genes define two distinct subfamilies, while the more homologous B genes represent different copies of the same gene type. The A.L11 and B.L11 introns are much longer, in each case because of a single inserted DNA segment that is missing from A.L12 or B.L12. The 2.1 X 10(3) base insertion in A.L11 is the first retrovirus-like transposable element characterized in Bombyx mori. The very short 5' flanking sequences of A/B.L11 and A/B.L12 (277 and 276 base-pairs) are distinct as shown by hybridization but both recur in additional chorion gene pairs, forming two respective classes that are expressed during distinctly different developmental periods. The divergently transcribed genes of each pair, which border the same 5' flanking sequence, are expressed co-ordinately, during the same developmental period. Detailed comparisons of the 5' flanking regions, and of the corresponding region of the Drosophila s15-1 chorion gene, revealed numerous, very short sequence elements that are shared. One such element, T-C-A-C-G-T, is also associated with all five sequenced Drosophila chorion genes. Some elements are repeated in a dyad symmetrical pattern, i.e. are associated with each of the two genes in a pair, while others, including T-C-A-C-G-T, occur only once per 5' flanking region, and, if functionally important, would presumably act bi-directionally on both genes of the pair.

Animals↗

The murine Wilms tumor suppressor gene (wt1) locus.

The Wilms tumor suppressor gene WT1 plays a crucial role in the etiology of various human diseases as well as in the development of specific organs including the kidneys, gonads and the spleen. At present the human as well as the Fugu wt1 locus have been characterized. We have used a PAC clone to analyze the murine wt1 locus and report here the structure of the wt1 gene as well as a characterization of the nine wt1 introns regarding their size and sequence at the exon/intron and intron/exon boundaries. In addition we provide a restriction map of the murine wt1 locus which should prove useful for the cloning of various constructs designed for the generation of mouse models. Prompted by the existence of a WT1 antisense transcript in humans we also examined strand-specific transcription at the murine wt1 locus. Our analysis suggests that there is no detectable antisense transcription of sequences within or immediately downstream of wt1 exon 1. We find, however, evidence for a divergent transcript which encompasses sequences at and around minor transcriptional initiation sites of wt1 and which is transcribed in the opposite direction. Despite the very high degree of similarity between the human and the murine wt1 sequence and expression as well as the presence of divergent transcripts in both cases, the existence of antisense transcription does not seem to be conserved between the two species.

Amino Acid Sequence↗

Demonstration of a divergent transcript from the bidirectional heavy chain immunoglobulin promoter VH441 in B-cells.

The mouse heavy chain immunoglobulin promoter VH441 can lead in vitro to bidirectional transcription, due to a symmetrical organization of immunoglobulin heavy chain promoters with two TATA-like sequences bracketing the upstream promoter element ATGCAAAT (the so called octamer). We demonstrate here that divergent transcription also occurs in vivo in mature B cells from a myeloma which expresses the VH441 gene and even from the spleen of BALB/c mice. The level of VH441 divergent transcript increases in the spleen of BALB/c mice after immunisation by beta-(1,6)-galactan, showing that it is expressed in B cells which actively transcribe the VH441 gene. The divergent transcript has been characterized: its major transcription start site was mapped within 33 base pairs from the divergent TATA-like region, it is unspliced and not polyadenylated. In the light of these results, the functions of the divergent transcript and the bidirectional promoter are discussed.

Animals↗

Cross-linking of Fc gamma receptor IIa and Fc gamma receptor IIIb induces different proadhesive phenotypes on human neutrophils.

Activation of polymorphonuclear leukocytes (PMN) plays an important role in vascular injury associated with systemic vasculitis and in models of autoantibody- and immune complex-mediated disease. The potential role of intravascular activation of PMN, however, is confounded by the observation that some stimuli injected i.v. (e.g., IL-8 and C5a) lead to L-selectin shedding by PMN, which inhibits attachment to endothelium and may be functionally anti-inflammatory. To explore the impact of Fc gamma receptor (Fc gamma R)-mediated activation on the PMN adhesive phenotype, Fc gamma RIIa (CD32) and Fc gamma RIIIb (Cd16) were targeted with receptor-specific reagents, and the expression of adhesion molecules-mediating rolling (L-selectin) and firm adhesion (CD11b/CD18) was measured. Engagement of either Fc gamma RIIa or Fc gamma RIIIb leads to activation, demonstrated by degranulation (upregulation of CD66b), and to increased expression of total CD11b/CD18 and functional CD11b/CD18 (I-domain). In contrast, L-selectin shedding induced by PMN Fc gamma R was divergent. Despite the 5- to 10-fold greater expression and engagement at saturation, activation via Fc gamma RIIIb led to little or no change in L-selectin expression. Stimulation of PMN with intact murine anti-receptor IgG1 showed a contribution of Fc gamma RIIa receptor polymorphisms, underscoring the direct influences of Fc gamma R allotypes on receptor function. These observations suggest that Fc gamma RIIIb-mediated activation of circulating PMN may lead to a proadhesive phenotype likely to promote systemic vascular damage. This Fc gamma R-mediated adhesive phenotype will vary with the receptors engaged and their allotypes, which, in turn, reflect properties of the immune complex and the genetics of the host.

Alleles↗

A role for Raf-1 in the divergent signaling pathways mediating insulin-stimulated glucose transport.

Downstream mediators of insulin signaling are thought to include multiple cytoplasmic serine/threonine kinases such as the product of the cellular proto-oncogene c-raf-1. To investigate a role for the Raf-1 protein kinase in insulin-stimulated glucose transport, a gene encoding an oncogenically activated Raf-1 mutant was introduced into 3T3-L1 fibroblasts by retroviral gene transfer. Expression of activated Raf-1 in differentiated 3T3-L1 adipocytes markedly increases hexose uptake compared to control adipocytes or those infected with the retroviral vector. Basal 2-deoxyglucose uptake in adipocytes expressing activated Raf-1 is approximately 40-fold higher than in parental adipocytes, and insulin further increases uptake about 1.2-fold. As determined by the plasma membrane "sheet" assay, Raf-1-expressing adipocytes contain greatly elevated levels of the ubiquitous glucose transporter (GLUT1) on the cell surface in the absence or presence of insulin. Total cellular GLUT1 protein is increased about 5-fold. In contrast, activated Raf-1 affects neither the expression of the "insulin-responsive" glucose transporter (GLUT4) nor its cellular distribution; GLUT4 is virtually undetectable on the plasma membrane in the absence of insulin and translocates normally following the addition of hormone. These data suggest that activation of Raf-1 mediates the chronic effect of insulin on hexose uptake but is not sufficient for the rapid translocation of GLUT4. Moreover, the differential effects of activated Raf-1 expression on the two transporter isoforms define divergent signaling pathways by which insulin regulates glucose transport in cultured adipocytes.

3T3 Cells↗