Elevated metabolites of acetaminophen in cord blood of children with obesity.

BACKGROUND: High-throughput metabolomics has been used cross-sectionally to evaluate differential metabolic profiles associated with human obesity. OBJECTIVES: This study longitudinally assessed the cord blood metabolome to explore if metabolic signatures of obesity at age 3-5 are apparent at birth. METHODS: In a nested case-control design, metabolomics analysis was performed on umbilical cord blood of 25 children who developed obesity by age 3-5 years, compared with 25 sex-matched non-obese children enrolled as part of an ongoing birth cohort. Logistic regression models were used to identify significant metabolites, adjusting for maternal pre-pregnancy obesity. RESULTS: Children who had obesity by age 3-5 years had elevated levels of medium and long chain fatty acids including stearate, oleate and palmitate at birth. Children with obesity were also more likely to have elevated levels of acetaminophen metabolites at birth, specifically: 3-(N-acetyl-L-cystein-S-yl) acetaminophen, 2-hydroxyacetaminophen sulfate, 2-methoxyacetaminophen glucuronide and p-acetamidophenyl glucuronide. CONCLUSION: Although the observed increases in lipids are consistent with previous metabolomic studies of obesity, this study is the first to report associations between acetaminophen metabolites and obesity in children; however, we lack mechanistic insights for this link. Larger human studies with longer follow-up and laboratory-controlled animal experiments are needed to clarify associations.

Immunolocalization of ecto-nucleoside triphosphate diphosphohydrolase 3 in rat brain: implications for modulation of multiple homeostatic systems including feeding and sleep-wake behaviors.

Three anti-peptide antisera were raised against three distinct amino acid sequences of ecto-nucleoside triphosphate diphosphohydrolase 3 (NTPDase3), characterized by Western blot analyses, and used to determine the distribution of NTPDase3 protein in adult rat brain. The three antisera all yielded similar immunolocalization data, leading to increased reliability of the results obtained. Unlike NTPDase1 and NTPDase2, NTPDase3 immunoreactivity was detected exclusively in neurons. Immunoreactivity was localized primarily to axon-like structures with prominent staining of presynaptic elements. Specific perikaryal immunostaining was detected primarily in scattered neurons near the lateral hypothalamic area and the perifornical nucleus. High densities of immunoreactive axon-like fibers were present in midline regions of the forebrain and midbrain. Highly scattered NTPDase3 positive fibers were observed in the cerebral cortex, the hippocampal formation, and the basal ganglia. Moreover, very high densities of immunostained fibers were detected in the mediobasal hypothalamus, with the overall mesencephalic pattern of staining associated closely with hormone responsive nuclei. High densities of NTPDase3 positive terminals were also associated with noradrenergic neurons. However, co-immunolocalization studies revealed clearly that NTPDase3 immunoreactivity was not localized within the noradrenaline cells or terminals. In contrast, nearly all of the NTPDase3 immunopositive hypothalamic cells, and most fibers in the mid- and hindbrain, also expressed hypocretin-1/orexin-A. The overall pattern of expression and co-localization with hypocretin-1/orexin-A suggests that NTPDase3, by regulating the extracellular turnover of ATP, may modulate feeding, sleep-wake, and other behaviors through diverse homeostatic systems.

Time course and manner of Purkinje neuron death following a single ethanol exposure on postnatal day 4 in the developing rat.

The present study was designed to evaluate the time course and manner of Purkinje cell death following a single ethanol dose delivered intragastrically on postnatal day (PN) 4 to rat pups. Analysis included immunolabeling of Purkinje cells with antibody specific for calbindin D28k and counting of Purkinje cells in each lobule of a mid-vermal slice. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling analysis and immunodetection for cleaved (activated) caspase-3 enzyme was used to identify apoptosis, with calbindin D28k co-immunolabeling to identify apoptotic Purkinje cells. Finally, immunodetection for cytochrome c, again with co-labeling using calbindin D28k antibody, identified intracellular release of cytochrome c from the mitochondria into the cytoplasm of Purkinje cells. The data demonstrate that a single dose of ethanol results in a significant and extensive, lobular dependent loss of Purkinje cells within 24 h after administration. Extensive loss in the early developing lobules (I-III, VIII-X) and less to no loss in the later developing lobules (IV-VII) is consistent with prior literature reports on the ethanol-induced effects on Purkinje cells at this age. Clear and consistent evidence of apoptotic Purkinje cells was identified and the pattern was transient in nature. Finally, cytochrome c is released from the mitochondria of Purkinje cells in a time course consistent with the activation of the mitochondrial pathway of apoptosis. These data support the hypothesis that ethanol-induced loss of Purkinje cells involves apoptotic mechanisms. Furthermore, the initiation of apoptosis by ethanol is consistent with ethanol-induced interruptions of Purkinje cell neurotrophic support leading to activation of the mitochondrial pathway of apoptosis.

Estrogenic actions in the brain: estrogen, phytoestrogens, and rapid intracellular signaling mechanisms.

The endogenous gonadal steroid 17beta-estradiol (E2) plays an important role in the development, maturation, and function of a wide variety of reproductive and nonreproductive tissues, including those of the nervous system. The actions of E2 at target tissues can be divided into 1) long-term "genomic" actions that are mediated by intracellular estrogen receptor-induced changes in gene expression and 2) rapid actions that modulate a diverse array of intracellular signal transduction cascades. Environmental estrogens are compounds present in the environment that can mimic, and in some cases antagonize, the effects of endogenous estrogens. As a result of these actions, there is currently much interest within the scientific community regarding the relative benefits or threats associated with exposure to different environmental estrogens. Within the general public there is considerable acceptance of the benefits associated with increased use of "natural" estrogens as a component of a healthy diet and in postmenopausal women as an alternative to estrogen replacement therapies. First, this review will focus attention on the role of estrogens in the central nervous system by briefly discussing some of the known mechanisms through which estrogen's effects are mediated, focusing on rapid intracellular signaling mechanisms during neurodevelopment. Second, with the hope of bringing attention to an area of study that until recently has received little consideration, we will briefly discuss phytoestrogens and suggest that these compounds have the potential to influence rapid E2-induced mechanisms in the nervous system in ways that may result in modified brain functions.

Early postnatal ethanol exposure selectively decreases BDNF and truncated TrkB-T2 receptor mRNA expression in the rat cerebellum.

Binge-like ethanol exposure on postnatal day (PN) 4 induces a concentration dependent loss of Purkinje cells in the rat cerebellum. The mechanism of this ethanol-induced Purkinje cell vulnerability is not presently understood. Nevertheless, the specific timing of this vulnerability leads us to consider the neurotrophin system crucial to the regulation of neuronal development. Differentiation, maturation, and survival of Purkinje cells are shown to involve an intimate interaction between brain-derived nerve growth factor (BDNF) and neurotrophin-3 (NT3) acting primarily through their specific tyrosine-kinase (Trk) receptors. We believe that the specific ethanol vulnerability, and the timing of this vulnerability result from alterations in the BDNF-NT3 interplay. We hypothesize that disruption of TrkB and/or TrkC mediated neurotrophin communication is, in part, responsible for the ethanol-induced loss of Purkinje cells during development. The current study was undertaken to define the impact of ethanol exposure at the onset of ethanol vulnerability on the relative concentrations of mRNA encoding the neurotrophic factor receptors TrkB and TrkC. The reverse transcriptase (RT) polymerase chain reaction (PCR) amplification technique was used to identify the relative expression levels of mRNA specific to these receptors as well as the truncated TrkB receptor isoforms. We identify a specific decrease in overall TrkB receptor mRNA expression that is primarily a function of the TrkB-T2 receptor isoform. Concurrent decreases in mRNA specific to BDNF were also identified. No significant alterations to the expression of TrkC mRNA were found indicating that ethanol-exposure appears to act selectively on the BDNF communication system.

Simplified serum- and steroid-free culture conditions for high-throughput viability analysis of primary cultures of cerebellar granule neurons.

A serum- and steroid-free primary culture system was developed for the maintenance and automated analysis of cerebellar granule cell viability. Conventional poly-lysine coated 96-well tissue culture plates serve as a platform for growth, experimental manipulation and subsequent automated analysis of these primary cultured neurons. Cerebellar granule neurons were seeded at densities ranging from 2 x 10(4) to 1.25 x 10(6) cells/cm(2) and maintained in serum- and steroid-free culture conditions for 7 days. Viability was subsequently determined by the reduction of [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS), and the degree of cell death occurring over that period was determined by the release of lactate dehydrogenase (LDH). At appropriate cell densities, the results of the MTS reduction and LDH release assays were directly proportional to the initial number of cerebellar granule cells plated. Those results indicate that an initial cell density of 0.5 - 1.0 x 10(5) cells per well (0.32 cm(2)) was appropriate for simultaneous analysis with the MTS reduction and LDH release assays. Both assays were then used to demonstrate the utility of this model system for analysis of tert-butyl-hydroperoxide and hydrogen peroxide induced oxidative stress. Additionally, the MTS reduction assay was used to demonstrate that the NMDA-receptor selective antagonist MK-801 was neuroprotective against glutamate-mediated excitotoxicity. This study defines a powerful and flexible primary culture system for cerebellar neurons that is useful for high-throughput analysis of factors that influence neuronal viability.

Identification of a developmental gradient of estrogen receptor expression and cellular localization in the developing and adult female rat primary somatosensory cortex.

Immunohistochemistry was used to investigate the spatiotemporal distribution of estrogen receptor alpha and beta (ER alpha, ER beta) in the posteromedial barrel subfield (PMBS) of the cerebral cortex in developing and adult female rats. Counting of immunopositive cells in predefined areas from each layer of the PMBS showed that at PN3, ER alpha immunoreactivity (IR) was present in every cell, whereas ER beta-IR was not detected. At PN6, about 59% of the cells were ER alpha immunopositive and low levels of ER beta-IR were observed in scattered cells. At PN18 the proportion of ER alpha-IR cells decreased to 49%; however, ER beta-IR became widespread and was detected in 39% of cells. By PN25 only faint ER alpha-IR was observed and in the adults ER alpha-IR was not detected. In contrast, at PN25 and in adults, ER beta-IR was detected in about half the cells of the PMBS. Regarding the cellular localization of ER-IR, at PN3 an outside-in gradient of cytoplasmic to nuclear localization of ER alpha-IR was observed. At PN18 and in adults ER beta-IR was preferentially localized to the nucleus of principal neurons, and to the cytoplasm of small, stellate-shaped interneurons. Together, these observations reveal a developmental transition of ER expression in the PMBS; ER alpha is expressed during early development, ER alpha and ER beta are co-expressed at later developmental times, and only ER beta is expressed in adults. These changes in ER expression and localization suggest that ER alpha and ER beta may play important, but different roles in the formation and function of the PMBS region of the primary somatosensory cortex.

Estrogen receptor beta immunoreactivity in differentiating cells of the developing rat cerebellum.

Estrogen receptors (ER) play a significant role in the development of some regions of the mammalian brain. Recently, ER-beta (ERbeta) mRNA and protein were shown to be expressed in the rat cerebellum. In the present study, the ontogeny of ERbeta protein expression was examined in the rat cerebellum during postnatal development. Western blot analysis indicated that a single ERbeta-like immunoreactive species of approximately 55 kDa was present in protein lysates prepared from the cerebella of female and male Sprague-Dawley rat pups. Immunocytochemical analysis of cerebellar sections from the midline vermis revealed that during development, the expression of ERbeta varied with age and cell-type, but not sex. In the developing cerebellum, highest levels of ERbeta-immunoreactivity (IR) were detected in neurons during neurite growth, and in some glia during migration. Throughout the first postnatal week, ERbeta-IR was localized to differentiating granule cells in the external germinal layer and to migrating glia. Differentiating granule cells expressed detectable levels of ERbeta throughout development. In Purkinje cells, ERbeta-IR was first detected on postnatal day 6 (P6), with peak intensities of immunostaining coinciding with the initiation of axonal and dendritic growth that occurs between P7 and P8. Expression of ERbeta-IR remained high during maturation of Purkinje cell dendrites, and then decreased to a lower level maintained in the adult. From the third postnatal week, ERbeta-IR was also detected in the later developing Golgi, stellate, and basket neurons. These results suggest that ERbeta may play a role in growth-related mechanisms during differentiation of cerebellar neurons and glia.

Regulated expression of estrogen receptor alpha and beta mRNA in granule cells during development of the rat cerebellum.

A semi-quantitative RT-PCR approach was used to characterize expression of the mRNA encoding estrogen receptors in developing cerebellar granule cells of the rat. Evidence is presented for expression of both ERalpha and ERbeta transcripts in granule cells throughout the first 15 postnatal days. While transcripts encoding both ERalpha and ERbeta were expressed in granule cells, the relative levels of expression varied significantly during the first two postnatal weeks of cerebellar development. The ERalpha mRNA was expressed at the lowest level on the first day following birth; whereas expression of ERbeta was highest on that day. On the fourth postnatal day the expression of ERalpha increased, while there was a significant decrease in ERbeta expression. Between postnatal day 4 and 15, the expression of the mRNA of each receptor varied in a similar fashion; expression decreased slightly between days 4 and 10 and then increased significantly on day 15. Alternative splicing of the ERbeta transcripts was also investigated and was likewise found to vary during granule cell development. Initially, the mRNA encoding the beta1 isoform was predominant, but by day 4, the beta2 isoform was the major isoform expressed. On postnatal days 7 and 10, there was not a significant difference between the level of beta1 and beta2 expressed. By day 15, beta1 was again the predominant ERbeta isoform accounting for nearly 90% of all ERbeta transcripts expressed.

Mutant alleles of the MRS2 gene of yeast nuclear DNA suppress mutations in the catalytic core of a mitochondrial group II intron.

Previous studies show that some yeast strains carrying point mutations of domain 5 that block splicing of a mitochondrial group II intron yield spontaneous revertants in which splicing is partially restored by dominant mutations of nuclear genes. Here we cloned and sequenced the suppressor allele of one such gene, and found it to be a missense mutation of the MRS2 gene (MRS2-L232F). The MRS2 gene was first implicated in group II intron splicing by the finding that overexpression of the wild-type gene weakly suppresses the splicing defect of a mutation of another intron. Tetrad analysis showed that independently isolated suppressors of two other domain 5 mutations are also allelles of the MRS2 gene and DNA sequencing identified a new missense mutation in each strain (MRS2-T230I and MRS2-L213M). All three suppressor mutations cause a temperature-sensitive respiration defect that is dominant negative in heterozygous diploids, but those strains splice the mutant intron at the elevated temperature. The three mutations are in a domain of the protein that is likely to be a helix-turn-helix region, so that effects of the mutations on protein-protein interactions may contribute to these phenotypes. These mutations suppress the splicing defect of many, but not all, of the available splicing defective mutations of aI5gamma, including mutations of several intron domains. Protein and RNA blot experiments show that the level of the protein encoded by the MRS2 gene, but not the mRNA, is elevated by these mutations. Interestingly, overexpression of the wild-type protein restores much lower levels of splicing than were obtained with similar elevated levels of the mutated Mrs2 proteins. The splicing phenotypes of these strains suggest a direct role for Mrs2 protein on group II intron splicing, but an indirect effect is not yet ruled out.

High-affinity kainate-type ion channels in rat cerebellar granule cells.

1. Patch-clamp recordings were made from rat cerebellar granule cells in primary culture. In cells pre-exposed to concanavalin A (ConA) to remove kainate receptor desensitization, concentration-response data for kainate showed two components. The EC50 value for the high-affinity component (4 microM) was consistent with activation of kainate-type channels. ConA enhanced the apparent potency of the kainate receptor ligand SYM 2081 by 100-fold. 2. In ConA-treated granule cells, currents evoked by 10 microM kainate were not significantly reduced by the AMPA receptor antagonist GYKI 53655, nor were these currents significantly reduced by the co-application of 100 microM AMPA. Currents activated by low concentrations of kainate in the presence of AMPA were completely inhibited by 10 microM La3+. 3. Single-cell reverse transcriptase-polymerase chain reaction (RT-PCR) analysis indicated that granule cells express both unedited (Q) and edited (R) versions of GluR5, with the majority of the GluR5 transcripts being unedited. In contrast, BluR6(R) was detected in seven cells and GluR6(Q) was detected in one granule cell. 4. Whole-cell current-voltage curves for kainate-type currents in granule cells were measured and the ratio of the slope conductances at +40 MV and -40 mV was used as an index of rectification. The mean +40 mV/-40 mV ratio determined from thirty-six granule cells was 1.3 +/- 0.1. Spectral density analysis of kainate-evoked whole-cell current noise gave values for the apparent single-channel conductance, gamma(noise), that were on average about 1 pS. 5. To compare further the properties of recombinant kainate channels with the native kainate-type channels in granule cells, we determined EC50 and gamma(noise) values for SYM 2081 in stable cell lines expressing either (GluR6(R) or GluR6(R) and KA2. Co-expression of KA2 with GluR6(R) shifts the EC50 and gamma(noise) values determined for SYM 2081 closer to the values typically found for native kainate-type channels in granule cells. 6. The results demonstrate that cerebellar granule cells in culture express functional kainate-type channels and that in most cells these channels show properties that are similar to those determined for heteromeric channels formed from GluR6(R) and KA2. However, the results also suggest that different granule cells express different repertoires of kainate-type channels with different, and perhaps variable, subunit composition.

Characterization of RNA editing of the glutamate-receptor subunits GluR5 and GluR6 in granule cells during cerebellar development.

The non-NMDA class of ionotropic glutamate receptors are subject to RNA editing resulting in single amino acid changes within individual subunits that make up these oligomeric receptors. These amino acid changes result in significant alterations of important channel properties. Both edited and unedited versions of the kainate-receptor subunits GluR5 and GluR6 are present in brain, but whether this reflects the expression of both versions in individual types of neurons or differences in editing between different cell types is unclear. To characterize editing in a single identified type of central neuron, we have determined the extent to which GluR5 and GluR6 mRNAs are edited in acutely isolated cerebellar granule cells. RT-PCR analysis revealed that editing at each site in GluR5 and GluR6 increased during early postnatal development. The Q/R site was predominantly unedited in GluR5, whereas GluR6 was mostly edited. The Q/R and Y/C sites of GluR6 were edited to similar extents, whereas a smaller percentage of transcripts were edited at the I/V site. The expression of two double-stranded RNA adenosine deaminases implicated in GluR editing (DRADA and RED1) increased in granule cells between postnatal days 1 and 15. Finally, cerebellar granule cells express a previously unreported variant of RED1 which appears to arise from developmentally regulated alternative splicing.

Cloning of the cDNA encoding the sodium channel beta 1 subunit from rabbit.

Here, we report the nucleotide (nt) sequence of the cDNA encoding the sodium channel beta 1 subunit from rabbit (o beta 1). Cloning of the o beta 1 cDNA was accomplished by reverse transcription-polymerase chain reaction using rabbit brain RNA as a template for cDNA synthesis. The nt sequence of the o beta 1 cDNA predicts a 218-amino-acid polypeptide which is 96.3 and 97.3% identical to the beta 1 from human (h beta 1) and rat (r beta 1), respectively.

Cloning of a sodium channel alpha subunit from rabbit Schwann cells.

Overlapping cDNA clones spanning the entire coding region of a Na-channel alpha subunit were isolated from cultured Schwann cells from rabbits. The coding region predicts a polypeptide (Nas) of 1984 amino acids exhibiting several features characteristic of Na-channel alpha subunits isolated from other tissues. Sequence comparisons showed that the Nas alpha subunit resembles most the family of Na channels isolated from brain (approximately 80% amino acid identity) and is least similar (approximately 55% amino acid identity) to the atypical Na channel expressed in human heart and the partial rat cDNA, NaG. As for the brain II and III isoforms, two variants of Nas exist that appear to arise by alternative splicing. The results of reverse transcriptase-polymerase chain reaction experiments suggest that expression of Nas transcripts is restricted to cells in the peripheral and central nervous systems. Expression was detected in cultured Schwann cells, sciatic nerve, brain, and spinal cord but not in skeletal or cardiac muscle, liver, kidney, or lung.

Studies of point mutants define three essential paired nucleotides in the domain 5 substructure of a group II intron.

Domain 5 (D5) is a highly conserved, largely helical substructure of group II introns that is essential for self-splicing. Only three of the 14 base pairs present in most D5 structures (A2.U33, G3.U32, and C4.G31) are nearly invariant. We have studied effects of point mutations of those six nucleotides on self-splicing and in vivo splicing of aI5 gamma, an intron of the COXI gene of Saccharomyces cerevisiae mitochondria. Though none of the point mutations blocked self-splicing under one commonly used in vitro reaction condition, the most debilitating mutations were at G3 and G4. Following mitochondrial Biolistic transformation, it was found that mutations at A2, G3, and C4 blocked respiratory growth and splicing while mutations at the other sites had little effect on either phenotype. Intra-D5 second-site suppressors showed that pairing between nucleotides at positions 2 and 33 and 4 and 31 is especially important for D5 function. At the G3.U32 wobble pair, the mutant A.U pair blocks splicing, but a revertant of that mutant that can form an A+.C base pair regains some splicing. A dominant nuclear suppressor restores some splicing to the G3A mutant but not the G3U mutant, suggesting that a purine is required at position 3. These findings are discussed in terms of the hypothesis of Madhani and Guthrie (H. D. Madhani and C. Guthrie, Cell 71:803-817, 1992) that helix 1 formed between yeast U2 and U6 small nuclear RNAs may be the spliceosomal cognate of D5.

The responsiveness of a tetracycline-sensitive expression system differs in different cell lines.

A tetracycline-sensitive inducible expression system was used to regulate the expression of neurotransmitter receptor genes in two mammalian cell lines. The dopamine D3-receptor was stably expressed in GH3 cells, and GluR6 (a glutamate receptor subunit) was stably expressed in human embryonic kidney (HEK 293) cells. Three striking differences were found. 1) In the inactive state, virtually no D3-receptor expression was found in GH3 cells, whereas substantial levels of GluR6 expression were found in HEK 293 cells. 2) The induction of expression obtained upon removal of tetracycline was robust in GH3 cells but only modest in HEK 293 cells. 3) Whereas in each clonal cell line, the expression of a co-transfected hybrid transactivator is clearly regulated in a tetracycline-responsive manner, in the induced state, its mRNA levels were found to be very low in GH3 cells and very high in HEK 293 cells. The results indicate that, in contrast to GH3 cells, HEK 293 cells do not provide a cellular environment in which the expression of a heterologous gene can be tightly controlled in a tetracycline-responsive manner.

Splicing defective mutants of the COXI gene of yeast mitochondrial DNA: initial definition of the maturase domain of the group II intron aI2.

Six mutations blocking the function of a seven intron form of the mitochondrial gene encoding subunit I of cytochrome c oxidase (COXI) and mapping upstream of exon 3 were isolated and characterized. A cis-dominant mutant of the group IIA intron 1 defines a helical portion of the C1 substructure of domain 1 as essential for splicing. A trans-recessive mutant confirms that the intron 1 reading frame encodes a maturase function. A cis-dominant mutant in exon 2 was found to have no effect on the splicing of intron 1 or 2. A trans-recessive mutant, located in the group IIA intron 2, demonstrates for the first time that intron 2 encodes a maturase. A genetic dissection of the five missense mutations present in the intron 2 reading frame of that strain demonstrates that the maturase defect results from one or both of the missense mutations in a newly-recognized conserved sequence called domain X.

Mutation of the conserved first nucleotide of a group II intron from yeast mitochondrial DNA reduces the rate but allows accurate splicing.

Group II introns have a phylogenetically conserved 5'-terminal pentanucleotide, -G1U2G3C4G5-, that resembles the conserved 5' end sequence of nuclear pre-mRNA introns. No functional interaction or catalytic role for the conserved G1 position has been proposed, although a tertiary structure involving -G3C4- has been implicated in splicing in vitro. We have analyzed splicing phenotypes both in vitro and in vivo for all three point mutants affecting guanosine at position 1 (G1) of intron 5 gamma from the COXI gene of yeast mitochondrial DNA. While all of these G1N substitutions slow splicing in vitro, G1C is clearly the most defective. All three mutant transcripts splice as accurately as the wild-type transcript, although the yield of lariat intron is reduced. The branched trinucleotide core includes the mutated position 1 nucleotide linked to the canonical branchpoint adenosine. The mutant lariats vary significantly in their susceptibility to the debranching activity from human cells. After wild-type, G1A was most sensitive, G1U was somewhat resistant, while G1C was highly resistant to debranching. These mutant lariats had normal ribozyme activity for promoting spliced exon reopening. The three mutant introns were transformed into otherwise normal yeast mitochondrial DNA. These mutants grow on nonfermentable carbon sources and splce aI5 gamma to yield excised intron lariat and mRNA. Nonetheless, each mutant splices with reduced efficiency, roughly parallel to their in vitro activity. In vivo, all three mutants accumulate both the pre-mRNA retaining intron 5 gamma and the lariat splicing intermediate containing intron and 3' exon. Clearly, this primary sequence element, shared with nuclear pre-mRNA introns, has a very different functional significance in group II splicing.

A maturase-encoding group IIA intron of yeast mitochondria self-splices in vitro.

Intron 1 of the coxI gene of yeast mitochondrial DNA (aI1) is a group IIA intron that encodes a maturase function required for its splicing in vivo. It is shown here to self-splice in vitro under some reaction conditions reported earlier to yield efficient self-splicing of group IIB introns of yeast mtDNA that do not encode maturase functions. Unlike the group IIB introns, aI1 is inactive in 10 mM Mg2+ (including spermidine) and requires much higher levels of Mg2+ and added salts (1M NH4Cl or KCl or 2M (NH4)2SO4) for ready detection of splicing activity. In KCl-stimulated reactions, splicing occurs with little normal branch formation; a post-splicing reaction of linear excised intron RNA that forms shorter lariat RNAs with branches at cryptic sites was evident in those samples. At low levels of added NH4Cl or KCl, the precursor RNA carries out the first reaction step but appears blocked in the splicing step. AI1 RNA is most reactive at 37-42 degrees C, as compared with 45 degrees C for the group IIB introns; and it lacks the KCl- or NH4Cl-dependent spliced-exon reopening reaction that is evident for the self-splicing group IIB introns of yeast mitochondria. Like the group IIB intron aI5 gamma, the domain 4 of aI1 can be largely deleted in cis, without blocking splicing; also, trans-splicing of half molecules interrupted in domain 4 occurs. This is the first report of a maturase-encoding intron of either group I or group II that self-splices in vitro.