Gene number in an invertebrate chordate, Ciona intestinalis

Gene number can be considered a pragmatic measure of biological complexity, but reliable data is scarce. Estimates for vertebrates are 50-100,000 genes per haploid genome, whereas invertebrate estimates fall below 25,000. We wished to test the hypothesis that the origin of vertebrates coincided with extensive gene creation. A prediction is that gene number will differ sharply between invertebrate and vertebrate members of the chordate phylum. A gene number estimation method requiring limited sequence sampling of genomic DNA was developed and validated by using data for Caenorhabditis elegans. Using the method, we estimated that the invertebrate chordate Ciona intestinalis has 15,500 protein-coding genes (±3,700). This number is significantly lower than gene numbers of vertebrate chordates, but similar to those of invertebrates in distantly related phyla. The data indicate that evolution of vertebrates was accompanied by a dramatic increase in protein-coding capacity of the genome.

Tropomyosin directly modulates actomyosin mechanical performance at the level of a single actin filament

Muscle contraction is the result of myosin cross-bridges (XBs) cyclically interacting with the actin-containing thin filament. This interaction is modulated by the thin filament regulatory proteins, troponin and tropomyosin (Tm). With the use of an in vitro motility assay, the role of Tm in myosin’s ability to generate force and motion was assessed. At saturating myosin surface densities, Tm had no effect on thin filament velocity. However, below 50% myosin saturation, a significant reduction in actin–Tm filament velocity was observed, with complete inhibition of movement occurring at 12.5% of saturating surface densities. Under similar conditions, actin filaments alone demonstrated no reduction in velocity. The effect of Tm on force generation was assessed at the level of a single thin filament. In the absence of Tm, isometric force was a linear function of the density of myosin on the motility surface. At 50% myosin surface saturation, the presence of Tm resulted in a 2-fold enhancement of force relative to actin alone. However, no further potentiation of force was observed with Tm at saturating myosin surface densities. These results indicate that, in the presence of Tm, the strong binding of myosin cooperatively activates the thin filament. The inhibition of velocity at low myosin densities and the potentiation of force at higher myosin densities suggest that Tm can directly modulate the kinetics of a single myosin XB and the recruitment of a population of XBs, respectively. At saturating myosin conditions, Tm does not appear to affect the recruitment or the kinetics of myosin XBs.

Restoration of β-adrenergic signaling in failing cardiac ventricular myocytes via adenoviral-mediated gene transfer

Cardiovascular gene therapy is a novel approach to the treatment of diseases such as congestive heart failure (CHF). Gene transfer to the heart would allow for the replacement of defective or missing cellular proteins that may improve cardiac performance. Our laboratory has been focusing on the feasibility of restoring β-adrenergic signaling deficiencies that are a characteristic of chronic CHF. We have now studied isolated ventricular myocytes from rabbits that have been chronically paced to produce hemodynamic failure. We document molecular β-adrenergic signaling defects including down-regulation of myocardial β-adrenergic receptors (β-ARs), functional β-AR uncoupling, and an up-regulation of the β-AR kinase (βARK1). Adenoviral-mediated gene transfer of the human β2-AR or an inhibitor of βARK1 to these failing myocytes led to the restoration of β-AR signaling. These results demonstrate that defects present in this critical myocardial signaling pathway can be corrected in vitro using genetic modification and raise the possibility of novel inotropic therapies for CHF including the inhibition of βARK1 activity in the heart.

Effect of reduced myristoylated alanine-rich C kinase substrate expression on hippocampal mossy fiber development and spatial learning in mutant mice: Transgenic rescue and interactions with gene background

The myristoylated alanine-rich C kinase substrate (MARCKS) is a prominent protein kinase C (PKC) substrate in brain that is expressed highly in hippocampal granule cells and their axons, the mossy fibers. Here, we examined hippocampal infrapyramidal mossy fiber (IP-MF) limb length and spatial learning in heterozygous Macs mutant mice that exhibit an ≈50% reduction in MARCKS expression relative to wild-type controls. On a 129B6(N3) background, the Macs mutation produced IP-MF hyperplasia, a significant increase in hippocampal PKCɛ expression, and proficient spatial learning relative to wild-type controls. However, wild-type 129B6(N3) mice exhibited phenotypic characteristics resembling inbred 129Sv mice, including IP-MF hypoplasia relative to inbred C57BL/6J mice and impaired spatial-reversal learning, suggesting a significant contribution of 129Sv background genes to wild-type and possibly mutant phenotypes. Indeed, when these mice were backcrossed with inbred C57BL/6J mice for nine generations to reduce 129Sv background genes, the Macs mutation did not effect IP-MF length or hippocampal PKCɛ expression and impaired spatial learning relative to wild-type controls, which now showed proficient spatial learning. Moreover, in a different strain (B6SJL(N1), the Macs mutation also produced a significant impairment in spatial learning that was reversed by transgenic expression of MARCKS. Collectively, these data indicate that the heterozygous Macs mutation modifies the expression of linked 129Sv gene(s), affecting hippocampal mossy fiber development and spatial learning performance, and that MARCKS plays a significant role in spatial learning processes.

Long-distance transcriptional enhancement by the histone acetyltransferase PCAF

Enhancers are defined by their ability to stimulate gene activity from remote sites and their requirement for promoter-proximal upstream activators to activate transcription. Here we demonstrate that recruitment of the p300/CBP-associated factor PCAF to a reporter gene is sufficient to stimulate promoter activity. The PCAF-mediated stimulation of transcription from either a distant or promoter-proximal position depends on the presence of an upstream activator (Sp1). These data suggest that acetyltransferase activity may be a primary component of enhancer function, and that recruitment of polymerase and enhancement of transcription are separable. Transcriptional activation by PCAF requires both its acetyltransferase activity and an additional activity within its N terminus. We also show that the simian virus 40 enhancer and PCAF itself are sufficient to counteract Mad-mediated repression. These results are compatible with recent models in which gene activity is regulated by the competition between deacetylase-mediated repression and enhancer-mediated recruitment of acetyltransferases.

Activation of transcription factor AP-2 mediates UVA radiation- and singlet oxygen-induced expression of the human intercellular adhesion molecule 1 gene

UVA radiation is the major component of the UV solar spectrum that reaches the earth, and the therapeutic application of UVA radiation is increasing in medicine. Analysis of the cellular effects of UVA radiation has revealed that exposure of human cells to UVA radiation at physiological doses leads to increased gene expression and that this UVA response is primarily mediated through the generation of singlet oxygen. In this study, the mechanisms by which UVA radiation induces transcriptional activation of the human intercellular adhesion molecule 1 (ICAM-1) were examined. UVA radiation was capable of inducing activation of the human ICAM-1 promoter and increasing ICAM-1 mRNA and protein expression. These UVA radiation effects were inhibited by singlet oxygen quenchers, augmented by enhancement of singlet oxygen life-time, and mimicked in unirradiated cells by a singlet oxygen-generating system. UVA radiation as well as singlet oxygen-induced ICAM-1 promoter activation required activation of the transcription factor AP-2. Accordingly, both stimuli activated AP-2, and deletion of the putative AP-2-binding site abrogated ICAM-1 promoter activation in this system. This study identified the AP-2 site as the UVA radiation- and singlet oxygen-responsive element of the human ICAM-1 gene. The capacity of UVA radiation and/or singlet oxygen to induce human gene expression through activation of AP-2 indicates a previously unrecognized role of this transcription factor in the mammalian stress response.

Enhancement of induced disease resistance by simultaneous activation of salicylate- and jasmonate-dependent defense pathways in Arabidopsis thaliana

The plant-signaling molecules salicylic acid (SA) and jasmonic acid (JA) play an important role in induced disease resistance pathways. Cross-talk between SA- and JA-dependent pathways can result in inhibition of JA-mediated defense responses. We investigated possible antagonistic interactions between the SA-dependent systemic acquired resistance (SAR) pathway, which is induced upon pathogen infection, and the JA-dependent induced systemic resistance (ISR) pathway, which is triggered by nonpathogenic Pseudomonas rhizobacteria. In Arabidopsis thaliana, SAR and ISR are effective against a broad spectrum of pathogens, including the foliar pathogen Pseudomonas syringae pv. tomato (Pst). Simultaneous activation of SAR and ISR resulted in an additive effect on the level of induced protection against Pst. In Arabidopsis genotypes that are blocked in either SAR or ISR, this additive effect was not evident. Moreover, induction of ISR did not affect the expression of the SAR marker gene PR-1 in plants expressing SAR. Together, these observations demonstrate that the SAR and the ISR pathway are compatible and that there is no significant cross-talk between these pathways. SAR and ISR both require the key regulatory protein NPR1. Plants expressing both types of induced resistance did not show elevated Npr1 transcript levels, indicating that the constitutive level of NPR1 is sufficient to facilitate simultaneous expression of SAR and ISR. These results suggest that the enhanced level of protection is established through parallel activation of complementary, NPR1-dependent defense responses that are both active against Pst. Therefore, combining SAR and ISR provides an attractive tool for the improvement of disease control.

Gene-experience interaction alters the cholinergic septohippocampal pathway of mice

Spatial learning requires the septohippocampal pathway. The interaction of learning experience with gene products to modulate the function of a pathway may underlie use-dependent plasticity. The regulated release of nerve growth factor (NGF) from hippocampal cultures and hippocampus, as well as its actions on cholinergic septal neurons, suggest it as a candidate protein to interact with a learning experience. A method was used to evaluate NGF gene-experience interaction on the septohippocampal neural circuitry in mice. The method permits brain region-specific expression of a new gene by using a two-component approach: a virus vector directing expression of cre recombinase; and transgenic mice carrying genomic recombination substrates rendered transcriptionally inactive by a “floxed” stop cassette. Cre recombinase vector delivery into transgenic mouse hippocampus resulted in recombination in 30% of infected cells and the expression of a new gene in those cells. To examine the interaction of the NGF gene and experience, adult mice carrying a NGF transgene with a floxed stop cassette (NGFXAT) received a cre recombinase vector to produce localized unilateral hippocampal NGF gene expression, so-called “activated” mice. Activated and control nonactivated NGFXAT mice were subjected to different experiences: repeated spatial learning, repeated rote performance, or standard vivarium housing. Latency, the time to complete the learning task, declined in the repeated spatial learning groups. The measurement of interaction between NGF gene expression and experience on the septohippocampal circuitry was assessed by counting retrogradely labeled basal forebrain cholinergic neurons projecting to the hippocampal site of NGF gene activation. Comparison of all NGF activated groups revealed a graded effect of experience on the septohippocampal pathway, with the largest change occurring in activated mice provided with repeated learning experience. These data demonstrate that plasticity of the adult spatial learning circuitry can be robustly modulated by experience-dependent interactions with a specific hippocampal gene product.

DNase I-hypersensitive sites I and II of the human growth hormone locus control region are a major developmental activator of somatotrope gene expression

High-level expression of the human growth hormone (hGH) gene is limited to somatotrope and lactosomatotrope cells of the anterior pituitary. We previously identified a locus control region (LCR) for the hGH gene composed of four tissue-specific DNase I-hypersensitive sites (HS) located between −14.6 kb and −32 kb 5′ to the hGH transcription start site that is responsible for establishing a physiologically regulated chromatin domain for hGH transgene expression in mouse pituitary. In the present study we demonstrated that the LCR mediates somatotrope and lactosomatotrope restriction on an otherwise weakly and diffusely expressed hGH transgene. The subregion of the LCR containing the two pituitary-specific HS, HSI and HSII (−14.6 to −16.2 kb relative to the hGH promoter and denoted HSI,II), was found to be sufficient for mediating somatotrope and lactosomatotrope restriction, for appropriately timed induction of hGH transgene expression between embryonic days 15.5 and 16.5, and for selective extinction of hGH expression in mature lactotropes. When studied by cell transfection, the HSI,II fragment selectively enhanced transcription in a presomatotrope-derived cell line, although at levels (2- to 3-fold) well below that seen in vivo. The LCR activity of the HSI,II element was therefore localized by scoring transgene expression in fetal founder pituitaries at embryonic day 18.5. The data from these studies indicated that a 404-bp segment of the HSI,II region encodes a critical subset of LCR functions, including the establishment of a productive chromatin environment, cell-specific restriction and enhancement of expression, and appropriately timed induction of the hGH transgene during embryonic development.

The transcript release factor PTRF augments ribosomal gene transcription by facilitating reinitiation of RNA polymerase I

Termination of murine rDNA transcription by RNA polymerase I (Pol I) requires pausing of Pol I by terminator-bound TTF-I (transcription termination factor for Pol I), followed by dissociation of the ternary complex by PTRF (Pol I and transcript release factor). To examine the functional correlation between transcription termination and initiation, we have compared transcription on terminator-containing and terminator-less rDNA templates. We demonstrate that terminated RNA molecules are more efficiently synthesized than run-off transcripts, indicating that termination facilitates reinitiation. Transcriptional enhancement is observed in multiple- but not single-round transcription assays measuring either promoter-dependent or promoter-independent Pol I transcription. Increased synthesis of terminated transcripts is observed in crude extracts but not in a PTRF-free reconstituted transcription system, indicating that PTRF-mediated release of pre-rRNA is responsible for transcriptional enhancement. Consistent with PTRF serving an important role in modulating the efficiency of rRNA synthesis, PTRF exhibits pronounced charge heterogeneity, is phosphorylated at multiple sites and fractionates into transcriptionally active and inactive forms. The results suggest that regulation of PTRF activity may be an as yet unrecognized means to control the efficiency of ribosomal RNA synthesis.

Enhancement of translation by the epsilon element is independent of the sequence of the 460 region of 16S rRNA

The epsilon enhancer element is a pyrimidine-rich sequence that increases expression of T7 gene 10 and a number of Escherichia coli mRNAs during initiation of translation and inhibits expression of the recF mRNA during elongation. Based on its complementarity to the 460 region of 16S rRNA, it has been proposed that epsilon exerts its enhancer activity by base pairing to this complementary rRNA sequence. We have tested this model of enhancer action by constructing mutations in the 460 region of 16S rRNA and examining expression of epsilon-containing CAT reporter genes and recF–lacZ fusions in strains expressing the mutant rRNAs. Replacement of the 460 E.coli stem–loop with that of Salmonella enterica serovar Typhimurium or a stem–loop containing a reversal of all 8 bp in the helical region produced fully functional rRNAs with no apparent effect on cell growth or expression of any epsilon-containing mRNA. Our experiments confirm the reported effects of the epsilon elements on gene expression but show that these effects are independent of the sequence of the 460 region of 16S rRNA, indicating that epsilon–rRNA base pairing does not occur.

An evaluation of the performance of cDNA microarrays for detecting changes in global mRNA expression

The cDNA microarray is one technological approach that has the potential to accurately measure changes in global mRNA expression levels. We report an assessment of an optimized cDNA microarray platform to generate accurate, precise and reliable data consistent with the objective of using microarrays as an acquisition platform to populate gene expression databases. The study design consisted of two independent evaluations with 70 arrays from two different manufactured lots and used three human tissue sources as samples: placenta, brain and heart. Overall signal response was linear over three orders of magnitude and the sensitivity for any element was estimated to be 2 pg mRNA. The calculated coefficient of variation for differential expression for all non-differentiated elements was 12–14% across the entire signal range and did not vary with array batch or tissue source. The minimum detectable fold change for differential expression was 1.4. Accuracy, in terms of bias (observed minus expected differential expression ratio), was less than 1 part in 10 000 for all non-differentiated elements. The results presented in this report demonstrate the reproducible performance of the cDNA microarray technology platform and the methods provide a useful framework for evaluating other technologies that monitor changes in global mRNA expression.

Effect of nicotine on brain activation during performance of a working memory task

Nicotine influences cognition and behavior, but the mechanisms by which these effects occur are unclear. By using positron emission tomography, we measured cognitive activation (increases in relative regional cerebral blood flow) during a working memory task [2-back task (2BT)] in 11 abstinent smokers and 11 ex-smokers. Assays were performed both after administration of placebo gum and 4-mg nicotine gum. Performance on the 2BT did not differ between groups in either condition, and the pattern of brain activation by the 2BT was consistent with reports in the literature. However, in the placebo condition, activation in ex-smokers predominated in the left hemisphere, whereas in smokers, it occurred in the right hemisphere. When nicotine was administered, activation was reduced in smokers but enhanced in ex-smokers. The lateralization of activation as a function of nicotine dependence suggests that chronic exposure to nicotine or withdrawal from nicotine affects cognitive strategies used to perform the memory task. Furthermore, the lack of enhancement of activation after nicotine administration in smokers likely reflects tolerance.

Hydroperoxide lyase depletion in transgenic potato plants leads to an increase in aphid performance

Hydroperoxide lyases (HPLs) catalyze the cleavage of fatty acid hydroperoxides to aldehydes and oxoacids. These volatile aldehydes play a major role in forming the aroma of many plant fruits and flowers. In addition, they have antimicrobial activity in vitro and thus are thought to be involved in the plant defense response against pest and pathogen attack. An HPL activity present in potato leaves has been characterized and shown to cleave specifically 13-hydroperoxides of both linoleic and linolenic acids to yield hexanal and 3-hexenal, respectively, and 12-oxo-dodecenoic acid. A cDNA encoding this HPL has been isolated and used to monitor gene expression in healthy and mechanically damaged potato plants. HPL gene expression is subject to developmental control, being high in young leaves and attenuated in older ones, and it is induced weakly by wounding. HPL enzymatic activity, nevertheless, remains constant in leaves of different ages and also after wounding, suggesting that posttranscriptional mechanisms may regulate its activity levels. Antisense-mediated HPL depletion in transgenic potato plants has identified this enzyme as a major route of 13-fatty acid hydroperoxide degradation in the leaves. Although these transgenic plants have highly reduced levels of both hexanal and 3-hexenal, they show no phenotypic differences compared with wild-type ones, particularly in regard to the expression of wound-induced genes. However, aphids feeding on the HPL-depleted plants display approximately a two-fold increase in fecundity above those feeding on nontransformed plants, consistent with the hypothesis that HPL-derived products have a negative impact on aphid performance. Thus, HPL-catalyzed production of C6 aldehydes may be a key step of a built-in resistance mechanism of plants against some sucking insect pests.

Activation domain-mediated enhancement of activator binding to chromatin in mammalian cells.

DNA binding by transcriptional activators is typically an obligatory step in the activation of gene expression. Activator binding and subsequent steps in transcription are repressed by genomic chromatin. Studies in vitro have suggested that overcoming this repression is an important function of some activation domains. Here we provide quantitative in vivo evidence that the activation domain of GAL4-VP16 can increase the affinity of GAL4 for its binding site on genomic DNA in mammalian cells. Moreover, the VP16 activation domain has a much greater stimulatory effect on expression from a genomic reporter gene than on a transiently transfected reporter gene, where factor binding is more permissive. We found that not all activation domains showed a greater activation potential in a genomic context, suggesting that only some activation domains can function in vivo to alleviate the repressive effects of chromatin. These data demonstrate the importance of activation domains in relieving chromatin-mediated repression in vivo and suggest that one way they function is to increase binding of the activator itself.