Epigenetic regulation of flowering.

The acceleration of flowering by prolonged low temperature treatment (vernalization) has unique properties including the floral transition occurring at a time separate from the vernalization treatment. This implies the vernalization condition is inherited through mitotic divisions, but this vernalized state is not inherited from one generation to the next. FLC, the key gene mediating this response in the Arabidopsis is repressed by histone modifications involving the VRN2 protein complex. Other protein complexes participate in activating the gene. While many plant species depend on vernalization for optimising flowering time, the genes involved differ between dicot and monocot plants in both Arabidopsis and cereals, vernalization regulates photoperiod control of flowering by preventing the induction of the floral promoter FT by long days in autumn but allowing induction of FT in spring and hence flowering occurs at an optimal time in the annual life cycle.

UBIQUITIN-SPECIFIC PROTEASE 26 is required for seed development and the repression of PHERES1 in Arabidopsis.

The Arabidopsis mutant Atubp26 initiates autonomous endosperm at a frequency of approximately 1% in the absence of fertilization and develops arrested seeds at a frequency of approximately 65% when self-pollinated. These phenotypes are similar to those of the FERTILIZATION INDEPENDENT SEED (FIS) class mutants, mea, fis2, fie, and Atmsi1, which also show development of the central cell into endosperm in the absence of fertilization and arrest of the embryo following fertilization. Atubp26 results from a T-DNA insertion in the UBIQUITIN-SPECIFIC PROTEASE gene AtUBP26, which catalyzes deubiquitination of histone H2B and is required for heterochromatin silencing. The paternal copy of AtUBP26 is able to complement the loss of function of the maternal copy in postfertilization seed development. This contrasts to the fis class mutants where the paternal FIS copy does not rescue aborted seeds. As in the fis class mutants, the Polycomb group (PcG) complex target gene PHERES1 (PHE1) is expressed at higher levels in Atubp26 ovules than in wild type; there is a lower level of H3K27me3 at the PHE1 locus. The phenotypes suggest that AtUBP26 is required for normal seed development and the repression of PHE1.

Intramolecular heterogeneity of mitochondrial DNA of Drosophila melanogaster.

DNA from purified mitochondria of Drosophila melanogaster can be isolated as supercoiled molecules which when nicked have a contour length of 5.9 micron. Partial denaturation mapping shows regional heterogeneity of base composition with one early denaturing region, with a calculated GC content close to zero, extending over 20% of the genome. DNA isolated from unfertilized eggs shows nuclear and mitochondrial DNA in equal proportions; we found no evidence of other cytoplasmic species.

Molecular analysis of ds controlling element mutations at the adh1 locus of maize.

An active maize Adhl-F gene, a Ds-induced mutant of this gene, and two independent Ac-induced revertant alleles have been isolated. The Ds mutant differs from the progenitor allele in having a 405-base pair insertion flanked by a direct repeat of 8 bp. The repeat is a duplication of the 8 bp existing at the point of insertion in the 5' untranslated region of the gene. The insertion sequence is AT-rich (A, adenine; T, thymine) and has 11-bp inverted repeat sequences at its termini. In the revertants the insertion with its inverted repeats is deleted, but the 8-bp direct repeats remain in modified form. These results establish that the 405-bp sequence is a Ds element. The Adh1 messenger RNA level is low in the Ds mutant, and it appears that new sites for transcription initiation or RNA processing or both are used. There are at least 30 sequences in the maize genome related to the Ds element.

DNA methylation, a key regulator of plant development and other processes.

Recent research has demonstrated that DNA methylation plays an integral role in regulating the timing of flowering and in endosperm development. The identification of key genes controlling these processes, the expression of which is altered in plants with low methylation, opens the way to understanding how DNA methylation regulates plant development.

The control of flowering by vernalization.

The process by which vernalization, the exposure of a germinating seed or a juvenile plant to a prolonged period of low temperature, promotes flowering in the adult plant has remained a mystery for many years. The recent isolation of one of the key genes involved in vernalization, FLOWERING LOCUS C, has now provided an insight into the molecular mechanism involved, including the role of DNA methylation.

Anaerobically regulated aldolase gene of maize. A chimaeric origin?

The sequence of the anaerobically induced fructose 1,6-bisphosphate aldolase gene of maize is presented. Analysis of the upstream sequences of the aldolase gene reveals a six base-pair sequence (TGGTTT) with perfect homology to one of the sub-regions of the anaerobic regulatory element (ARE) which is responsible for the anaerobic induction of the maize alcohol dehydrogenase 1 gene (Adh1). In the aldolase gene this sequence is located at position -70 relative to the start of transcription, in a small segment proven by functional analysis to be important for expression of the aldolase gene. Since this six base-pair sequence has been shown to be critical for anaerobic induction of the Adh1 mRNA, is in the functional promoter region of aldolase and is also present in a homologous position in Adh2 (another anaerobically-induced gene), we suggest this hexanucleotide is essential for anaerobic regulation of each of these genes. The maize aldolase gene is about 50% homologous at the amino acid level to the animal aldolase gene but has a completely different intron/exon structure. While the rat aldolase gene has nine introns the maize gene has a single large intron near the N terminus of the coding region. Because there is 55% homology downstream from the intron and very little homology upstream, we suggest that the maize gene has acquired a 5' region containing signals for anaerobic regulation and fortuitously adding a new N-terminal region to the protein. We must suppose that the plant gene has lost the remaining introns.

Structure and expression of an alcohol dehydrogenase 1 gene from Pisum sativum (cv. "Greenfeast").

Three genomic clones for anaerobically inducible alcohol dehydrogenase (Adh) have been isolated from Pisum sativum cv. "Greenfeast" via cDNA cloning. One of these contains a complete gene, has exon sequences corresponding to one of the cDNA sequences and is likely to be an expressed gene. This gene has a structure similar to the Adh genes of maize, with introns in the same positions in the coding sequence but differing in their lengths and nucleotide sequences. At the nucleotide level the coding sequence is 75% homologous to both maize Adh1 and Adh2 and 80% homologous to the Adh gene from Arabidopsis, but has an extra coding triplet in exon 1 that is not found in the other plant Adh genes. The non-translated regions of all the gene transcripts are widely divergent between species. A short segment of the pea Adh promoter region (-290 to +57) was fused to a reporter gene and introduced into protoplasts of Nicotiana plumbaginifolia by electroporation. Transient expression of the introduced gene increased markedly when the transfected protoplasts were incubated under anaerobic conditions, showing that cis-acting regulatory signals necessary for anaerobic control of expression reside in the -290 to +57 segment. Sequence comparisons between this region and the corresponding regions of maize and Arabidopsis Adh genes have identified short sequences that may be involved in the anaerobic regulation of plant Adh genes.

Cloning of the Arabidopsis and rice formaldehyde dehydrogenase genes: implications for the origin of plant ADH enzymes.

This article reports the cloning of the genes encoding the Arabidopsis and rice class III ADH enzymes, members of the alcohol dehydrogenase or medium chain reductase/dehydrogenase superfamily of proteins with glutathione-dependent formaldehyde dehydrogenase activity (GSH-FDH). Both genes contain eight introns in exactly the same positions, and these positions are conserved in plant ethanol-active Adh genes (class P). These data provide further evidence that plant class P genes have evolved from class III genes by gene duplication and acquisition of new substrate specificities. The position of introns and similarities in the nucleic acid and amino acid sequences of the different classes of ADH enzymes in plants and humans suggest that plant and animal class III enzymes diverged before they duplicated to give rise to plant and animal ethanol-active ADH enzymes. Plant class P ADH enzymes have gained substrate specificities and evolved promoters with different expression properties, in keeping with their metabolic function as part of the alcohol fermentation pathway.

Sex chromosome meiotic drive systems in Drosophila melanogaster I. Abnormal spermatid development in males with a heterochromatin-deficient X chromosome (sc-4sc-8).

The meiotic drive chracteristics of the In(1)sc-4Lsc-8R/Y system have been examined by genetic analysis and by light and electron microscopy. sc-4sc8-/Y males show a direct correlation between nondisjunction frequency and meiotic drive. Temperature-shift experiments reveal that the temperature-sensitive period for nondisjunction is at meiosis, whereas that for meiotic drive has both meiotic and post-meiotic components. Cytological analyses in the light and electron microscopes reveal failures in spermiogenesis in the tests of sc-4sc-8 males. The extent of abnormal spermatid development increases as nondisjunction becomes more extreme.

Multiple meiotic drive systems in the Drosophila melanogaster male.

The behaviour of two "meiotic drive" systems, Segregation-Distorter (SD) and the sex chromosome sc(4)sc(8) has been examined in the same meiocyte. It has been found that the two systems interact in a specific way. When the distorting effects of SD and sc(4)sc(8) are against each other, there is no detectable interaction. Each system is apparently oblivious to the presence of the other, gametes being produced according to independence expectations. However when the affected chromosomes are at the same meiotic pole an interaction occurs; the survival probability of the gamete containing both distorted chromosomal products is increased, rather than being decreased by the combined action of two systems.

Two Alleles of Maize ALCOHOL DEHYDROGENASE 1 Have 3' Structural and Poly(a) Addition Polymorphisms.

Two standard electrophoretic alleles of the maize alcohol dehydrogenase 1 locus (Adh1-1S and Adh1-1F) have been isolated and characterized. Restriction endonuclease mapping shows that a region of less than 5 kb is conserved in both alleles and is flanked both 5' and 3' by regions highly polymorphic for restriction sites. Nucleotide sequence comparison of these two alleles reveals that polymorphism in the 3' flanking region is due to rearrangements including tandem duplications, a transposable element-like insertion and a deletion. S1 nuclease analysis shows that both the Adh1-1S and the Adh1-1F alleles contain multiple poly(A) addition sites; four sites are observed for the Adh1-1S alleles and seven sites for the Adh1-1F allele. Only two of these poly(A) addition sites appear to be identical in the two alleles. No consensus signal for poly(A) addition is observed near any of these sites.

Evidence for a role for AtMYB2 in the induction of the Arabidopsis alcohol dehydrogenase gene (ADH1) by low oxygen.

The transcription factor AtMYB2 binds to two sequence motifs in the promoter of the Arabidopsis ADH1 gene. The binding to the GT-motif (5'-TGGTTT-3') is essential for induction of ADH1 by low oxygen, while binding to the second motif, MBS-2, is not essential for induction. We show that AtMYB2 is induced by hypoxia with kinetics compatible with a role in the regulation of ADH1. Like ADH1, AtMYB2 has root-limited expression. When driven by a constitutive promoter, AtMYB2 is able to transactivate ADH1 expression in transient assays in both Arabidopsis and Nicotiana plumbaginifolia protoplasts, and in particle bombardment of Pisum sativum leaves. Mutation of the GT-motif abolished binding of AtMYB2 and caused loss of activity of the ADH1 promoter in both transient assays and transgenic Arabidopsis plants. These results are consistent with AtMYB2 being a key regulatory factor in the induction of the ADH1 promoter by low oxygen.

A TM3-like MADS-box gene from Eucalyptus expressed in both vegetative and reproductive tissues.

MADS-box genes in plants are a diverse class of transcription factors that are involved in regulating developmental processes, particularly meristem and organ identity during floral development. They are characterized by a highly conserved MADS-box domain of 59 amino acids that binds to specific DNA sequences. We report the characterization of a cDNA clone, ETL (Eucalyptus TM3 Like), from Eucalyptus globulus subspecies bicostata encoding a putative transcription factor of the MADS-box class that is strongly expressed in both vegetative and floral tissues, suggesting that it regulates processes other than floral development. The clone was isolated from a floral bud cDNA library with a probe generated from Eucalyptus genomic DNA by PCR using degenerate primers to the MADS-box of the floral regulatory gene APETALA 1. The ETL cDNA clone encodes a putative protein of 206 amino acids that contains an N-terminal MADS-box and a helical domain of approx. 60 amino acids predicted to form a coiled-coil (K-box). These structural features are characteristic of plant MADS-box proteins. The MADS-box domain contains all the signature residues of a class of MADS-box genes typified by the tomato gene TM3 and overall, ETL shows 56% amino acid identity to TM3. Like TM3, the ETL gene is expressed in both vegetative and reproductive organs, predominantly in root and shoot meristems and organ primordia, as well as in developing male and female floral organs.

Beneficial effect of siphoning in treatment of adult hydrocephalus.

OBJECTIVE: To increase awareness about the treatment of adult patients with shunt-nonresponsive hydrocephalus--a state characterized by marked ventriculomegaly, low intracranial pressure, and a patent cerebrospinal fluid diversionary shunt. DESIGN: Retrospective analysis of hospital and outpatient records. PATIENTS: Four patients with symptomatic ventriculomegaly and patent ventriculoperitoneal shunts treated with a protocol of progressive ventricular hypotension induced by external cerebrospinal fluid drainage. RESULTS: Severe clinical manifestations exhibited by the patients, including parkinsonian features, Parinaud syndrome, and extensor posturing, completely reversed once a normalization of ventricular size was achieved. External ventricular drainage pressures as low as -30 cm H2O were required to reduce ventricular size. All patients finally received a shunt incorporating a standard medium differential pressure valve with no antisiphon device. CONCLUSIONS: Shunt siphoning may be an essential mechanism by which cerebrospinal fluid shunting is effective in many patients with adult hydrocephalus. Cerebrospinal fluid shunts that contain an antisiphon device are ineffective in these patients, despite the attainment of "physiologic" intracranial pressures. Based on reported experimental and clinical evidence, it seems that the cause of this condition may be related to abnormally high intracranial compliance.

Surgical treatment of intractable neonatal-onset seizures: the role of positron emission tomography.

We have performed positron emission tomography (PET) with 2-deoxy-2[18F]fluoro-D-glucose (FDG) in eight infants and children (aged 18 days to 5 years) with medically refractory epilepsy of neonatal onset. It was hypothesized that in at least some of these infants a surgical approach (focal resection, cerebral hemispherectomy) might be of benefit in achieving seizure control, and that PET might assist in surgical selection. In three of the eight subjects, interictal PET revealed unilateral diffuse hypometabolism; following cerebral hemispherectomy in these three patients, all seizures ceased and there were no adverse effects. In one child, ictal PET showed hypermetabolism in the left frontal cortex, left striatum, and right cerebellum; a partial left cerebral hemispherectomy guided by intraoperative electrocorticography was performed, following which all seizures ceased. One infant had relative hypermetabolism in the right temporal and occipital lobes, right thalamus, and left frontal lobe on ictal PET, and EEG telemetry revealed a right occipitotemporal epileptic focus; this infant died from anesthetic complications following right occipitotemporal cortical resection. Of the three unoperated patients, one is a potential candidate for right frontal lobectomy, but the other two were not considered to be surgical candidates due to bilateral epileptogenicity. Neuropathologic correlation in our series revealed that PET is a sensitive test capable of detecting cytoarchitectural disturbances whereas CT and MRI failed in this regard. In addition, PET provides a very unique and important assessment of the functional integrity of brain regions outside the area of potential resection.

Hippocampal GABA and glutamate transporter immunoreactivity in patients with temporal lobe epilepsy.

OBJECTIVE: Sodium-coupled transporters remove extracellular neurotransmitters and alterations in their function could enhance or suppress synaptic transmission and seizures. This study determined hippocampal gamma-aminobutyric acid (GABA) and glutamate transporter immunoreactivity (IR) in temporal lobe epilepsy (TLE) patients. METHODS: Hippocampal sclerosis (HS) patients (n = 25) and non-HS cases (mass lesion and cryptogenic; n = 20) were compared with nonseizure autopsies (n = 8). Hippocampal sections were studied for neuron densities along with IR for glutamate decarboxylase (GAD; presynaptic GABA terminals), GABA transporter-1 (GAT-1; presynaptic GABA transporter), GAT-3 (astrocytic GABA transporter), excitatory amino acid transporter 3 (EAAT3; postsynaptic glutamate transporter), and EAAT2-1 (glial glutamate transporters). RESULTS: Compared with autopsies, non-HS cases with similar neuron counts showed: 1) increased GAD IR gray values (GV) in the fascia dentata outer molecular layer (OML), hilus, and stratum radiatum; 2) increased GAT-1 OML GVs; 3) increased astrocytic GAT-3 GVs in the hilus and Ammon's horn; and 4) no IR differences for EAAT3-1. HS patients with decreased neuron densities demonstrated: 1) increased OML and inner molecular layer GAD puncta; 2) decreased GAT-1 puncta relative to GAD in the stratum granulosum and pyramidale; 3) increased GAT-1 OML GVs; 4) decreased GAT-3 GVs; 5) increased EAAT3 IR on remaining granule cells and pyramids; 6) decreased glial EAAT2 GVs in the hilus and CA1 stratum radiatum associated with neuron loss; and 7) increased glial EAAT1 GVs in CA2/3 stratum radiatum. CONCLUSIONS: Hippocampal GABA and glutamate transporter IR differ in TLE patients compared with autopsies. These data support the hypothesis that excitatory and inhibitory neurotransmission and seizure susceptibility could be altered by neuronal and glial transporters in TLE patients.

Aberrant hippocampal mossy fiber sprouting correlates with greater NMDAR2 receptor staining.

This study determined in temporal lobe epilepsy patients and rats injected with intrahippocampal kainate (KA) whether fascia dentata molecular layer mossy fiber sprouting was associated with increases in NMDAR2 immunoreactivity (IR). Patients with hippocampal sclerosis (n = 11) were compared with those with temporal mass lesions (n = 7) and material obtained at autopsies (n = 4); and unilateral KA-injected rat hippocampi (n = 7) were compared with the contralateral saline-injected side and non-lesioned animals (n = 7; control). Hippocampi were studied for neo-Timm's stained mossy fiber sprouting and NMDAR2 IR. The staining was quantified as gray values (GV) using computer image analysis. Hippocampal sclerosis patients and KA-injected rats showed the greatest inner molecular layer (IML) mossy fiber sprouting and NMDAR2 staining. Compared with autopsies and patients with mass lesions, hippocampal sclerosis patients had greater IML neo-Timm's (p = 0.0018) and NMDAR2 staining (p = 0.0063). Similarly, compared with controls and saline-injected rats, KA-injected hippocampi showed greater IML mossy fiber sprouting and NMDAR2 IR (p = 0.0001). Furthermore, IML mossy fiber sprouting positively correlated with greater IML NMDAR2 staining in both human and experimental rat groups (p < 0.0099). These results support the hypothesis that in severely damaged hippocampi abnormal mossy fiber sprouting and concordant increases in IML NMDAR2 receptor staining may contribute or partially explain granule cell hyperexcitability and the pathophysiology of hippocampal epilepsy.

Neuroamine related compounds in the CSF of hydrocephalic rabbits.

The effects of neonatal hydrocephalus on the levels of tyrosine, tryptophan, 5-hydroxyindoleacetic acid (5-HIAA), and homovanillic acid (HVA) in CSF were determined by high-performance liquid chromatography (HPLC) with fluorometric detection in normal and chronically hydrocephalic rabbits. The hydrocephalic rabbits showed a highly significant increase in both the serotonin metabolite 5-HIAA and the dopamine metabolite HVA. There were no significant effects of the hydrocephalus on either tyrosine or tryptophan levels. There was a significant positive correlation between the intracranial pressure (ICP) and the increase in 5-HIAA and HVA, but not with the two precursor amino acids. There was a significant decrease in these amino acid precursors with age in both groups. A trend towards higher levels of 5-HIAA and HVA in older rabbits was also evident, however this change was not to the degree found in the hydrocephalics. These data indicate that increased ICP affects the mechanism of removal of 5-HIAA and HVA from the cerebrospinal fluid.

Neurophysiology of neocortical slices resected from children undergoing surgical treatment for epilepsy.

The recent emergence of surgical treatment of childhood epilepsy has led to the accessibility of young human cerebral tissue for electrophysiological studies of the mechanisms involved in epileptogenesis. Intracellular recordings were obtained from neurons in slices prepared from neocortical tissue resected from children (3 months to 15 years) with catastrophic epilepsy. Data from 'least abnormal' versus 'most abnormal' tissue were compared; the evaluation of the degree of abnormality was based on several clinical criteria. Hypotheses concerning NMDA receptors, local synaptic circuits, and epileptiform bursts were tested. The NMDA receptor-mediated component of synaptic responses, which was isolated pharmacologically, had a voltage dependence that was functionally mature by 8-10 months of age and did not appear to be altered even in the most abnormal tissue. Local inhibitory and excitatory synaptic circuits were present as early as 11 months and 8 months, respectively. Local excitatory circuits were sufficiently extensive in young children to initiate and sustain epileptiform activity when synaptic inhibition was suppressed. Bicuculline-induced epileptiform bursts were similar to those in adult human or animal neocortical slices. Burst duration and the presence of after-discharges were unrelated to patient age or tissue abnormality. These data demonstrate that (1) the electrophysiological properties of human neocortical neurons are very similar to those observed in animal experiments, (2) the mechanisms of neuronal communication are qualitatively mature within the first year of life, and (3) synaptic transmission and local neuronal circuits appear qualitatively normal, even in the most abnormal tissue from children with catastrophic epilepsy.