Gene cloning, sequence analysis, and expression of 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase

The gene encoding 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase (MHPCO; EC 1.14.12.4) was cloned by using an oligonucleotide probe corresponding to the N terminus of the enzyme to screen a DNA library of Pseudomonas sp. MA-1. The gene encodes for a protein of 379 amino acid residues corresponding to a molecular mass of 41.7 kDa, the same as that previously estimated for MHPCO. MHPCO was expressed in Escherichia coli and found to have the same properties as the native enzyme from Pseudomonas sp. MA-1. This study shows that MHPCO is a homotetrameric protein with one flavin adenine dinucleotide bound per subunit. Sequence comparison of the enzyme with other hydroxylases reveals regions that are conserved among aromatic flavoprotein hydroxylases.

A serotonin transporter gene intron 2 polymorphic region, correlated with affective disorders, has allele-dependent differential enhancer-like properties in the mouse embryo

Polymorphic regions consisting of a variable number of tandem repeats within intron 2 of the gene coding for the serotonin transporter protein 5-HTT have been associated with susceptibility to affective disorders. We have cloned two of these intronic polymorphisms, Stin2.10 and Stin2.12, into an expression vector containing a heterologous minimal promoter and the bacterial LacZ reporter gene. These constructs were then used to produce transgenic mice. In embryonic day 10.5 embryos, both Stin2.10 and Stin2.12 produced consistent β-galactosidase expression in the embryonic midbrain, hindbrain, and spinal cord floor plate. However, we observed that the levels of β-galactosidase expression produced by both the Stin2.10 and Stin2.12 within the rostral hindbrain differed significantly at embryonic day 10.5. Our data suggest that these polymorphic variable number of tandem repeats regions act as transcriptional regulators and have allele-dependent differential enhancer-like properties within an area of the hindbrain where the 5-HTT gene is known to be transcribed at this stage of development.

Targeted gene deletion of heme oxygenase 2 reveals neural role for carbon monoxide

Neuronal nitric oxide synthase (nNOS) generates NO in neurons, and heme-oxygenase-2 (HO-2) synthesizes carbon monoxide (CO). We have evaluated the roles of NO and CO in intestinal neurotransmission using mice with targeted deletions of nNOS or HO-2. Immunohistochemical analysis demonstrated colocalization of nNOS and HO-2 in myenteric ganglia. Nonadrenergic noncholinergic relaxation and cyclic guanosine 3′,5′ monophosphate elevations evoked by electrical field stimulation were diminished markedly in both nNOSΔ/Δ and HO-2Δ/Δ mice. In wild-type mice, NOS inhibitors and HO inhibitors partially inhibited nonadrenergic noncholinergic relaxation. In nNOSΔ/Δ animals, NOS inhibitors selectively lost their efficacy, and HO inhibitors were inactive in HO-2Δ/Δ animals.

Expression of the Recessive Glomerulosclerosis Gene Mpv17 Regulates MMP-2 Expression in Fibroblasts, the Kidney, and the Inner Ear of Mice

The recessive mouse mutant Mpv17 is characterized by the development of early-onset glomerulosclerosis, concomitant hypertension, and structural alterations of the inner ear. The primary cause of the disease is the loss of function of the Mpv17 protein, a peroxisomal gene product involved in reactive oxygen metabolism. In our search of a common mediator exerting effects on several aspects of the phenotype, we discovered that the absence of the Mpv17 gene product causes a strong increase in matrix metalloproteinase 2 (MMP-2) expression. This was seen in the kidney and cochlea of Mpv17-negative mice as well as in tissue culture cells derived from these animals. When these cells were transfected with the human Mpv17 homolog, an inverse causal relationship between Mpv17 and MMP-2 expression was established. These results indicate that the Mpv17 protein plays a crucial role in the regulation of MMP-2 and suggest that enhanced MMP-2 expression might mediate the mechanisms leading to glomerulosclerosis, inner ear disease, and hypertension in this model.

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.

Expression of a Hox gene, Cnox-2, and the division of labor in a colonial hydroid

We report the isolation and expression of the Hox gene, Cnox-2, in Hydractinia symbiolongicarpus, a hydrozoan displaying division of labor. We found different patterns of aboral-to-oral Cnox-2 expression among polyp polymorphs, and we show that experimental conversion of one polyp type to another is accompanied by concordant alteration in Cnox-2 expression. Our results are consistent with the suggestion that polyp polymorphism, characteristic of hydractiniid hydroids, arose via evolutionary modification of proportioning of head to body column.

t(11;22)(q23;q11.2) in acute myeloid leukemia of infant twins fuses MLL with hCDCrel, a cell division cycle gene in the genomic region of deletion in DiGeorge and velocardiofacial syndromes

We examined the MLL genomic translocation breakpoint in acute myeloid leukemia of infant twins. Southern blot analysis in both cases showed two identical MLL gene rearrangements indicating chromosomal translocation. The rearrangements were detectable in the second twin before signs of clinical disease and the intensity relative to the normal fragment indicated that the translocation was not constitutional. Fluorescence in situ hybridization with an MLL-specific probe and karyotype analyses suggested t(11;22)(q23;q11.2) disrupting MLL. Known 5′ sequence from MLL but unknown 3′ sequence from chromosome band 22q11.2 formed the breakpoint junction on the der(11) chromosome. We used panhandle variant PCR to clone the translocation breakpoint. By ligating a single-stranded oligonucleotide that was homologous to known 5′ MLL genomic sequence to the 5′ ends of BamHI-digested DNA through a bridging oligonucleotide, we formed the stem–loop template for panhandle variant PCR which yielded products of 3.9 kb. The MLL genomic breakpoint was in intron 7. The sequence of the partner DNA from band 22q11.2 was identical to the hCDCrel (human cell division cycle related) gene that maps to the region commonly deleted in DiGeorge and velocardiofacial syndromes. Both MLL and hCDCrel contained homologous CT, TTTGTG, and GAA sequences within a few base pairs of their respective breakpoints, which may have been important in uniting these two genes by translocation. Reverse transcriptase-PCR amplified an in-frame fusion of MLL exon 7 to hCDCrel exon 3, indicating that an MLL-hCDCrel chimeric mRNA had been transcribed. Panhandle variant PCR is a powerful strategy for cloning translocation breakpoints where the partner gene is undetermined. This application of the method identified a region of chromosome band 22q11.2 involved in both leukemia and a constitutional disorder.

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.

Inactivation of the cyclin-dependent kinase inhibitor p27 upon loss of the tuberous sclerosis complex gene-2

Tuberous sclerosis is an autosomal dominant disorder characterized by the development of aberrant growths in many tissues and organs. Linkage analysis revealed two disease-determining genes on chromosome 9 and chromosome 16. The tuberous sclerosis complex gene-2 (TSC2) on chromosome 16 encodes the tumor suppressor protein tuberin. We have shown earlier that loss of TSC2 is sufficient to induce quiescent cells to enter the cell cycle. Here we show that TSC2-negative fibroblasts exhibit a shortened G1 phase. Although the expression of cyclin E, cyclin A, p21, or Cdc25A is unaffected, TSC2-negative cells express much lower amounts of the cyclin-dependent kinase (CDK) inhibitor p27 because of decreased protein stability. In TSC2 mutant cells the amount of p27 bound to CDK2 is diminished, accompanied with elevated kinase activity. Ectopic expression studies revealed that the aforementioned effects can be reverted by transfecting TSC2 in TSC2-negative cells. High ectopic levels of p27 have cell cycle inhibitory effects in TSC2-positive cells but not in TSC2-negative counterparts, although the latter still depend on CDK2 activity. Loss of TSC2 induces soft agar growth of fibroblasts, a process that cannot be inhibited by high levels of p27. Both phenotypes of TSC2-negative cells, their resistance to the activity of ectopic p27, and the instability of endogenous p27, could be explained by our observation that the nucleoprotein p27 is mislocated into the cytoplasm upon loss of TSC2. These findings provide insights into the molecular mechanism of how loss of TSC2 induces cell cycle entry and allow a better understanding of its tumor suppressor function.

Embryo-Specific Gene Expression in Microspore-Derived Embryos of Brassica napus. An Interaction between Abscisic Acid and Jasmonic Acid1, 2

The induction of napin and oleosin gene expression in Brassica napus microspore-derived embryos (MDEs) was studied to assess the possible interaction between abscisic acid (ABA) and jasmonic acid (JA). Napin and oleosin transcripts were detected sooner following treatment with ABA than JA. Treatment of MDEs with ABA plus JA gave an additive accumulation of both napin and oleosin mRNA, the absolute amount being dependent on the concentration of each hormone. Endogenous ABA levels were reduced by 10-fold after treatment with JA, negating the possibility that the observed additive interaction was due to JA-induced ABA biosynthesis. Also, JA did not significantly increase the uptake of [3H-ABA] from the medium into MDEs. This suggests that the additive interaction was not due to an enhanced carrier-mediated ABA uptake by JA. Finally, when JA was added to MDEs that had been treated with the ABA biosynthesis inhibitor fluridone, napin mRNA did not increase. Based on these results with the MDE system, it is possible that embryos of B. napus use endogenous JA to modulate ABA effects on expression of both napin and oleosin. In addition, JA could play a causal role in the reduction of ABA that occurs during late stages of seed development.

A missense mutation of the Na+ channel αII subunit gene Nav1.2 in a patient with febrile and afebrile seizures causes channel dysfunction

Generalized epilepsy with febrile seizures plus (GEFS+), a clinical subset of febrile seizures (FS), is characterized by frequent episodes beyond 6 years of age (FS+) and various types of subsequent epilepsy. Mutations in β1 and αI-subunit genes of voltage-gated Na+ channels have been associated with GEFS+1 and 2, respectively. Here, we report a mutation resulting in an amino acid exchange (R187W) in the gene encoding the α-subunit of neuronal voltage-gated Na+ channel type II (Nav1.2) in a patient with FS associated with afebrile seizures. The mutation R187W occurring on Arg187, a highly conserved residue among voltage-gated Na+ channels, was not found in 224 alleles of unaffected individuals. Whole-cell patch clamp recordings on human embryonic kidney (HEK) cells expressing a rat wild-type (rNav1.2) and the corresponding mutant channels showed that the mutant channel inactivated more slowly than wild-type whereas the Na+ channel conductance was not affected. Prolonged residence in the open state of the R187W mutant channel may augment Na+ influx and thereby underlie the neuronal hyperexcitability that induces seizure activity. Even though a small pedigree could not show clear cosegregation with the disease phenotype, these findings strongly suggest the involvement of Nav1.2 in a human disease and propose the R187W mutation as the genetic defect responsible for febrile seizures associated with afebrile seizures.

Genetic complexity of pathogen perception by plants: The example of Rcr3, a tomato gene required specifically by Cf-2

Genetic analysis of plant–pathogen interactions has demonstrated that resistance to infection is often determined by the interaction of dominant plant resistance (R) genes and dominant pathogen-encoded avirulence (Avr) genes. It was postulated that R genes encode receptors for Avr determinants. A large number of R genes and their cognate Avr genes have now been analyzed at the molecular level. R gene loci are extremely polymorphic, particularly in sequences encoding amino acids of the leucine-rich repeat motif. A major challenge is to determine how Avr perception by R proteins triggers the plant defense response. Mutational analysis has identified several genes required for the function of specific R proteins. Here we report the identification of Rcr3, a tomato gene required specifically for Cf-2-mediated resistance. We propose that Avr products interact with host proteins to promote disease, and that R proteins “guard” these host components and initiate Avr-dependent plant defense responses.