Genetic integration of a nuclear -encoded mitochondrial gene with cardiac function

Cardiovascular disease (CVD) is the leading cause of death in the United States. One manifestation of CVD known to increase mortality is an enlarged, or hypertrophic heart. Hypertrophic cardiomyocytes adapt to increased contractile demand at the genetic level with a re-emergence of the fetal gene program and a downregulation of fatty acid oxidation genes with concomitant increased reliance on glucose-based metabolism. To understand the transcriptional regulatory pathways that implement hypertrophic directives we analyzed the upstream promoter region of the muscle specific isoform of the nuclear-encoded mitochondrial gene, carnitine palmitoyltransferase-1β (CPT-1β) in cultured rat neonatal cardiac myocytes. This enzyme catalyzes the rate-limiting step of fatty acid entry into β-oxidation and is downregulated in cardiac hypertrophy and failure, making it an attractive model for the study of hypertrophic gene regulation and metabolic adaptations. We demonstrate that the muscle-enriched transcription factors GATA-4 and SRF synergistically activate CPT-1β; moreover, DNA binding to cognate sites and intact protein structure are required. This mechanism coordinates upregulation of energy generating processes with activation of the energy consuming contractile promoter for cardiac α-actin. We hypothesized that fatty acid or glucose responsive transcription factors may also regulate CPT-1β. Oleate weakly stimulates CPT-1β activity; in contrast, the glucose responsive Upstream Stimulatory Factors (USF) dramatically depresses the CPT-1β reporter. USF regulates CPT-1β through a novel physical interaction with the cofactor PGC-1 and abrogation of MEF2A/PGC-1 synergistic stimulation. In this way, USF can inversely regulate metabolic gene programs and may play a role in the shift of metabolic substrate preference seen in hypertrophy. Failing hearts have elevated expression of the nuclear hormone receptor COUP-TF. We report that COUP-TF significantly suppresses reporter transcription independent of DNA binding and specific interactions with GATA-4, Nkx2.5 or USF. In summary, CPT-1β transcriptional regulation integrates mitochondrial gene expression with two essential cardiac functions: contraction and metabolic substrate oxidation. ^

Mendel’s dwarfing gene: cDNAs from the Le alleles and function of the expressed proteins

The major gibberellin (GA) controlling stem elongation in pea (Pisum sativum L.) is GA1, which is formed from GA20 by 3β-hydroxylation. This step, which limits GA1 biosynthesis in pea, is controlled by the Le locus, one of the original Mendelian loci. Mutations in this locus result in dwarfism. We have isolated cDNAs encoding a GA 3β-hydroxylase from lines of pea carrying the Le, le, le-3, and led alleles. The cDNA sequences from le and le-3 each contain a base substitution resulting in single amino acid changes relative to the sequence from Le. The cDNA sequence from led, a mutant derived from an le line, contains both the le “mutation” and a single-base deletion, which causes a shift in reading frame and presumably a null mutation. cDNAs from each line were expressed in Escherichia coli. The expression product for the clone from Le converted GA9 to GA4, and GA20 to GA1, with Km values of 1.5 μM and 13 μM, respectively. The amino acid substitution in the clone from le increased Km for GA9 100-fold and reduced conversion of GA20 to almost nil. Expression products from le and le-3 possessed similar levels of 3β-hydroxylase activity, and the expression product from led was inactive. Our results suggest that the 3β-hydroxylase cDNA is encoded by Le. Le transcript is expressed in roots, shoots, and cotyledons of germinating pea seedlings, in internodes and leaves of established seedlings, and in developing seeds.

Suppression of an absolute defect in Type IV pilus biogenesis by loss-of-function mutations in pilT, a twitching motility gene in Neisseria gonorrhoeae

Type IV pili of Neisseria gonorrhoeae, the Gram-negative etiologic agent of gonorrhea, facilitate colonization of the human host. Gonococcal PilT, a protein belonging to a large family of molecules sharing a highly conserved nucleotide binding domain motif, has been shown to be dispensable for organelle biogenesis but essential for twitching motility and competence for genetic transformation. Here, we show that the defect in pilus biogenesis resulting from mutations in the pilC gene, encoding a putative pilus-associated adhesin for human tissue, can be suppressed by the absence of functional PilT. These data conclusively demonstrate that PilT influences the Type IV pilus biogenesis pathway and strongly suggest that organelle expression is a dynamic process. In addition, these findings imply that PilT antagonizes the process of organelle biogenesis and provide the basis for a model for how the counteractive roles of PilT and PilC might relate mechanistically to the phenomenon of twitching motility.

Slow skeletal troponin I gene transfer, expression, and myofilament incorporation enhances adult cardiac myocyte contractile function

The functional significance of the developmental transition from slow skeletal troponin I (ssTnI) to cardiac TnI (cTnI) isoform expression in cardiac myocytes remains unclear. We show here the effects of adenovirus-mediated ssTnI gene transfer on myofilament structure and function in adult cardiac myocytes in primary culture. Gene transfer resulted in the rapid, uniform, and nearly complete replacement of endogenous cTnI with the ssTnI isoform with no detected changes in sarcomeric ultrastructure, or in the isoforms and stoichiometry of other myofilament proteins compared with control myocytes over 7 days in primary culture. In functional studies on permeabilized single cardiac myocytes, the threshold for Ca2+-activated contraction was significantly lowered in adult cardiac myocytes expressing ssTnI relative to control values. The tension–Ca2+ relationship was unchanged from controls in primary cultures of cardiac myocytes treated with adenovirus containing the adult cardiac troponin T (TnT) or cTnI cDNAs. These results indicate that changes in Ca2+ activation of tension in ssTnI-expressing cardiac myocytes were isoform-specific, and not due to nonspecific functional changes resulting from overexpression of a myofilament protein. Further, Ca2+-activated tension development was enhanced in cardiac myocytes expressing ssTnI compared with control values under conditions mimicking the acidosis found during myocardial ischemia. These results show that ssTnI enhances contractile sensitivity to Ca2+ activation under physiological and acidic pH conditions in adult rat cardiac myocytes, and demonstrate the utility of adenovirus vectors for rapid and efficient genetic modification of the cardiac myofilament for structure/function studies in cardiac myocytes.

Identification of bdm-1, a gene involved in G protein β-subunit function and α-subunit accumulation

Targeted disruption of Gα and Gβ genes has established the requirement of an intact G protein signaling pathway for optimal execution of several important physiological processes, including pathogenesis, in the chestnut blight fungus Cryphonectria parasitica. We now report the identification of a G protein signal transduction component, beta disruption mimic factor-1, BDM-1. Disruption of the corresponding gene, bdm-1, resulted in a phenotype indistinguishable from that previously observed after disruption of the Gβ subunit gene, cpgb-1. The BDM-1 deduced amino acid sequence contained several significant clusters of identity with mammalian phosducin, including a domain corresponding to a highly conserved 11-amino acid stretch that has been implicated in binding to the Gβγ dimer and two regions of defined Gβ/phosducin contact points. Unlike the negative regulatory function proposed for mammalian phosducin, the genetic data presented in this report suggest that BDM-1 is required for or facilitates Gβ function. Moreover, disruption of either bdm-1 or cpgb-1 resulted in a significant, posttranscriptional reduction in the accumulation of CPG-1, a key Gα subunit required for a range of vital physiological processes.

The Drosophila LIM-only gene, dLMO, is mutated in Beadex alleles and might represent an evolutionarily conserved function in appendage development

The process of wing patterning involves precise molecular mechanisms to establish an organizing center at the dorsal–ventral boundary, which functions to direct the development of the Drosophila wing. We report that misexpression of dLMO, a Drosophila LIM-only protein, in specific patterns in the developing wing imaginal disc, disrupts the dorsal–ventral (D-V) boundary and causes errors in wing patterning. When dLMO is misexpressed along the anterior–posterior boundary, extra wing outgrowth occurs, similar to the phenotype seen when mutant clones lacking Apterous, a LIM homeodomain protein known to be essential for normal D-V patterning of the wing, are made in the wing disc. When dLMO is misexpressed along the D-V boundary in third instar larvae, loss of the wing margin is observed. This phenotype is very similar to the phenotype of Beadex, a long-studied dominant mutation that we show disrupts the dLMO transcript in the 3′ untranslated region. dLMO normally is expressed in the wing pouch of the third instar wing imaginal disc during patterning. A mammalian homolog of dLMO is expressed in the developing limb bud of the mouse. This indicates that LMO proteins might function in an evolutionarily conserved mechanism involved in patterning the appendages.

Loss of function of the tuberous sclerosis 2 tumor suppressor gene results in embryonic lethality characterized by disrupted neuroepithelial growth and development

Germline defects in the tuberous sclerosis 2 (TSC2) tumor suppressor gene predispose humans and rats to benign and malignant lesions in a variety of tissues. The brain is among the most profoundly affected organs in tuberous sclerosis (TSC) patients and is the site of development of the cortical tubers for which the hereditary syndrome is named. A spontaneous germline inactivation of the Tsc2 locus has been described in an animal model, the Eker rat. We report that the homozygous state of this mutation (Tsc2Ek/Ek) was lethal in mid-gestation (the equivalent of mouse E9.5–E13.5), when Tsc2 mRNA was highly expressed in embryonic neuroepithelium. During this period homozygous mutant Eker embryos lacking functional Tsc2 gene product, tuberin, displayed dysraphia and papillary overgrowth of the neuroepithelium, indicating that loss of tuberin disrupted the normal development of this tissue. Interestingly, there was significant intraspecies variability in the penetrance of cranial abnormalities in mutant embryos: the Long–Evans strain Tsc2Ek/Ek embryos displayed these defects whereas the Fisher 344 homozygous mutant embryos had normal-appearing neuroepithelium. Taken together, our data indicate that the Tsc2 gene participates in normal brain development and suggest the inactivation of this gene may have similar functional consequences in both mature and embryonic brain.

The interphotoreceptor retinoid binding protein gene in therian mammals: Implications for higher level relationships and evidence for loss of function in the marsupial mole

The subclass Theria of Mammalia includes marsupials (infraclass Metatheria) and placentals (infraclass Eutheria). Within each group, interordinal relationships remain unclear. One limitation of many studies is incomplete ordinal representation. Here, we analyze DNA sequences for part of exon 1 of the interphotoreceptor retinoid binding protein gene, including 10 that are newly reported, for representatives of all therian orders. Among placentals, the most robust clades are Cetartiodactyla, Paenungulata, and an expanded African clade that includes paenungulates, tubulidentates, and macroscelideans. Anagalida, Archonta, Altungulata, Hyracoidea + Perissodactyla, Ungulata, and the “flying primate” hypothesis are rejected by statistical tests. Among marsupials, the most robust clade includes all orders except Didelphimorphia. The phylogenetic placement of the monito del monte and the marsupial mole remains unclear. However, the marsupial mole sequence contains three frameshift indels and numerous stop codons in all three reading frames. Given that the interphotoreceptor retinoid binding protein gene is a single-copy gene that functions in the visual cycle and that the marsupial mole is blind with degenerate eyes, this finding suggests that phenotypic degeneration of the eyes is accompanied by parallel changes at the molecular level as a result of relaxed selective constraints.

Identification of partial loss of function p53 gene mutations utilizing a yeast-based functional assay

Missense mutations within the central DNA binding region of p53 are the most prevalent mutations found in human cancer. Numerous studies indicate that ‘hot-spot’ p53 mutants (which comprise ∼30% of human p53 gene mutations) are largely devoid of transcriptional activity. However, a growing body of evidence indicates that some non-hot-spot p53 mutants retain some degree of transcriptional activity in vivo, particularly against strong p53 binding sites. We have modified a previously described yeast-based p53 functional assay to readily identify such partial loss of function p53 mutants. We demonstrate the utility of this modified p53 functional assay using a diverse panel of p53 mutants.

Structure and function in rhodopsin: high level expression of a synthetic bovine opsin gene and its mutants in stable mammalian cell lines.

Stable mammalian cell lines harboring a synthetic bovine opsin gene have been derived from the suspension-adapted HEK293 cell line. The opsin gene is under the control of the immediate-early cytomegalovirus promoter/enhancer in an expression vector that also contains a selectable marker (Neo) governed by a relatively weak promoter. The cell lines expressing the opsin gene at high levels are selected by growth in the presence of high concentrations of the antibiotic geneticin. Under the conditions used for cell growth in suspension, opsin is produced at saturated culture levels of more than 2 mg/liter. After reconstitution with 11-cis-retinal, rhodopsin is purified to homogeneity in a single step by immunoaffinity column chromatography. Rhodopsin thus prepared (> 90% recovery at concentrations of up to 15 microM) is indistinguishable from rhodopsin purified from bovine rod outer segments by the following criteria: (i) UV/Vis absorption spectra in the dark and after photobleaching and the rate of metarhodopsin II decay, (ii) initial rates of transducin activation, and (iii) the rate of phosphorylation by rhodopsin kinase. Although mammalian cell opsin migrates slower than rod outer segment opsin on SDS/polyacrylamide gels, presumably due to a different N-glycosylation pattern, their mobilities after deglycosylation are identical. This method has enabled the preparation of several site-specific mutants of bovine opsin in comparable amounts.

Altered surfactant function and structure in SP-A gene targeted mice.

The surfactant protein A (SP-A) gene was disrupted by homologous recombination in embryonic stem cells that were used to generate homozygous SP-A-deficient mice. SP-A mRNA and protein were not detectable in the lungs of SP-A(-/-) mice, and perinatal survival of SP-A(-/-) mice was not altered compared with wild-type mice. Lung morphology, surfactant proteins B-D, lung tissue, alveolar phospholipid pool sizes and composition, and lung compliance in SP-A(-/-) mice were unaltered. At the highest concentration tested, surfactant from SP-A(-/-) mice produced the same surface tension as (+/+) mice. At lower concentrations, minimum surface tensions were higher for SP-A(-/-) mice. At the ultrastructural level, type II cell morphology was the same in SP-A(+/+) and (-/-) mice. While alveolar phospholipid pool sizes were unperturbed, tubular myelin figures were decreased in the lungs of SP-A(-/-) mice. A null mutation of the murine SP-A gene interferes with the formation of tubular myelin without detectably altering postnatal survival or pulmonary function.

A novel function for Myc: inhibition of C/EBP-dependent gene activation.

We have investigated the effect of the v-Myc oncoprotein on gene expression in myelomonocytic cells. We find that v-Myc dramatically down-regulates the expression of myelomonocytic-specific genes, such as the chicken mim-1 and lysozyme genes, both of which are known targets for C/EBP transcription factors. We present evidence that Myc downregulates these genes by inhibiting the function of C/EBP transcription factors. Detailed examination of the inhibitory mechanism shows that amino-terminal sequences of v-Myc, but not its DNA-binding domain, are required for the suppression of C/EBP-dependent transactivation. Our findings identify a new function for Myc and reveal a novel mechanism by which Myc affects the expression of other genes.

The angiotensin II type 2 (AT2) receptor antagonizes the growth effects of the AT1 receptor: gain-of-function study using gene transfer.

The type 1 angiotensin II (AT1) receptor is well characterized but the type 2 (AT2) receptor remains an enigma. We tested the hypothesis that the AT2 receptor can modulate the growth of vascular smooth muscle cells by transfecting an AT2 receptor expression vector into the balloon-injured rat carotid artery and observed that overexpression of the AT2 receptor attenuated neointimal formation. In cultured smooth muscle cells, AT2 receptor transfection reduced proliferation and inhibited mitogen-activated protein kinase activity. Furthermore, we demonstrated that the AT2 receptor mediated the developmentally regulated decrease in aortic DNA synthesis at the latter stages of gestation. These results suggest that the AT2 receptor exerts an antiproliferative effect, counteracting the growth action of AT1 receptor.