The mechanism of cyclic electron flow.

Apart from the canonical light-driven linear electron flow (LEF) from water to CO2, numerous regulatory and alternative electron transfer pathways exist in chloroplasts. One of them is the cyclic electron flow around Photosystem I (CEF), contributing to photoprotection of both Photosystem I and II (PSI, PSII) and supplying extra ATP to fix atmospheric carbon. Nonetheless, CEF remains an enigma in the field of functional photosynthesis as we lack understanding of its pathway. Here, we address the discrepancies between functional and genetic/biochemical data in the literature and formulate novel hypotheses about the pathway and regulation of CEF based on recent structural and kinetic information.

Maximal cyclic electron flow rate is independent of PGRL1 in Chlamydomonas.

Cyclic electron flow (CEF) is defined as a return of the reductants from the acceptor side of Photosystem I (PSI) to the pool of its donors via the cytochrome b6f. It is described to be complementary to the linear electron flow and essential for photosynthesis. However, despite many efforts aimed to characterize CEF, its pathway and its regulation modes remain equivocal, and its physiological significance is still not clear. Here we use novel spectroscopic to measure the rate of CEF at the onset of light in the green alga Chlamydomonas reinhardtii. The initial redox state of the photosynthetic chain or the oxygen concentration do not modify the initial maximal rate of CEF (60 electrons per second per PSI) but rather strongly influence its duration. Neither the maximal rate nor the duration of CEF are different in the pgrl1 mutant compared to the wild type, disqualifying PGRL1 as the ferredoxin-plastoquinone oxidoreductase involved in the CEF mechanism.

The peculiar NPQ regulation in the stramenopile Phaeomonas sp. challenges the xanthophyll cycle dogma.

In changing light conditions, photosynthetic organisms develop different strategies to maintain a fine balance between light harvesting, photochemistry, and photoprotection. One of the most widespread photoprotective mechanisms consists in the dissipation of excess light energy in the form of heat in the photosystem II antenna, which participates to the Non Photochemical Quenching (NPQ) of chlorophyll fluorescence. It is tightly related to the reversible epoxidation of xanthophyll pigments, catalyzed by the two enzymes, the violaxanthin deepoxidase and the zeaxanthin epoxidase. In Phaeomonas sp. (Pinguiophyte, Stramenopiles), we show that the regulation of the heat dissipation process is different from that of the green lineage: the NPQ is strictly proportional to the amount of the xanthophyll pigment zeaxanthin and the xanthophyll cycle enzymes are differently regulated. The violaxanthin deepoxidase is already active in the dark, because of a low luminal pH, and the zeaxanthin epoxidase shows a maximal activity under moderate light conditions, being almost inactive in the dark and under high light. This light-dependency mirrors the one of NPQ: Phaeomonas sp. displays a large NPQ in the dark as well as under high light, which recovers under moderate light. Our results pinpoint zeaxanthin epoxidase activity as the prime regulator of NPQ in Phaeomonas sp. and therefore challenge the deepoxidase-regulated xanthophyll cycle dogma.

Polymorphism analysis of JRK/JH8, the human homologue of mouse jerky, and description of a rare mutation in a case of CAE evolving to JME.

Disruption of the function of the mouse jerky gene by transgene insertion causes generalized recurrent seizures reminiscent of human idiopathic generalized epilepsy (IGE). A human homologue, JRK/JH8, has been cloned, which maps to 8q24, a chromosomal region associated with several forms of IGE. JRK/JH8 is, therefore, a candidate locus for at least some forms of IGE. We report corrected cDNA sequences and extended open reading frames for the mouse jerky and human JRK/JH8 genes, which add 48 amino acids to the N-terminus of the Jerky protein and which extends the region of homology with the N-terminal DNA-binding domain of the centromere-binding protein, CENP-B. Systematic sequencing of the coding region of the extended JRK/JH8 gene identified single nucleotide polymorphisms that define three haplotypes, which were used for association studies in patients with idiopathic generalized epilepsy. We report one subject with childhood absence epilepsy (CAE) that evolved to juvenile myoclonic epilepsy (JME) that has a unique de novo mutation that results in a non-conservative amino acid change at a potential protein glycosylation site. Familial analysis supports a causal role for this mutation in the disease.

B219/OB-R 5'-UTR and leptin receptor gene-related protein gene expression in mouse brain and placenta: tissue-specific leptin receptor promoter activity.

Leptin receptor (OB-R) splice variants either encode proteins with different 3' cytoplasmic domains or have different 5' untranslated regions (UTR), indicative of dual promoters. The B219/OB-R promoter transcribes only OB-R transcripts, whereas the OB-R/GRP promoter initiates transcription of both OB-R and another protein of unknown function, called the leptin receptor gene-related protein (OB-RGRP). We compared expression of B219/OB-R 5'-UTR and OB-RGRP mRNAs by in situ hybridization. We thus assessed, by inference, the contributions of the two promoters to the leptin receptor transcript pool, in murine brain or in placenta, a tissue with abundant leptin receptor mRNA. Expression of B219/OB-R 5'-UTR mRNA (and thus by inference B219/OB-R promoter activity) in brain was similar in both distribution and relative intensity to OB-R mRNA. OB-RGRP mRNA (and thus by inference OB-R/GRP promoter activity) was widely distributed in murine brain, with elevated expression in the hypothalamic regions that express the leptin receptor mRNA, including the paraventricular nucleus. B219/OB-R 5'-UTR mRNA, but not OB-RGRP mRNA, was upregulated in hypothalamus of obese ob/ob mice. In placenta, B219/OB-R 5'-UTR mRNA was restricted to the maternal interface, and transcription of both long and short leptin receptor splice variants in the main body of the tissue thus proceeds via the OB-R/GRP promoter, strongly indicative of tissue-specific promoter usage.

Identification of seven novel nucleotide variants in the hepatocyte nuclear factor-1alpha (TCF1) promoter region in MODY patients.

Maturity onset diabetes of the young (MODY) is a heterogeneous subtype of type II diabetes mellitus. To date, five MODY genes have been identified. Mutations in the hepatocyte nuclear factor-1alpha (HNF-1alpha) gene are associated with MODY3. In the present work, we implemented the HNF-1alpha promoter region in the screening of MODY-suspect patients and identified seven variants not detected in control subjects. The family was available for the -119delG variant, and segregration between MODY and the variant is observed. Most of these variants are located in highly conserved regions and may alter HNF-1alpha expression through binding alteration of nuclear factors or other mechanisms. We demonstrate by functional studies that the transcriptional activity of the -283A>C and -218T>C variant promoters were 30% and 70% of the wild type activity, respectively. These data suggest that HNF-1alpha promoter variants could be diabetogenic mutations, and emphasize that the accurate HNF-1alpha expression is important for the maintenance of normal pancreatic beta cell function.

Anatomy of a homeoprotein revealed by the analysis of human MODY3 mutations.

Hepatocyte nuclear factor 1alpha (HNF1alpha) is an atypical dimeric homeodomain-containing protein that is expressed in liver, intestine, stomach, kidney, and pancreas. Mutations in the HNF1alpha gene are associated with an autosomal dominant form of non-insulin-dependent diabetes mellitus called maturity-onset diabetes of the young (MODY3). More than 80 different mutations have been identified so far, many of which involve highly conserved amino acid residues among vertebrate HNF1alpha. In the present work, we investigated the molecular mechanisms by which MODY3 mutations could affect HNF1alpha function. For this purpose, we analyzed the properties of 10 mutants resulting in amino acid substitutions or protein truncation. Some mutants have a reduced protein stability, whereas others are either defective in the DNA binding or impaired in their intrinsic trans-activation potential. Three mutants, characterized by a complete loss of trans-activation, behave as dominant negatives when transfected with the wild-type protein. These data define a clear causative relationship between MODY3 mutations and functional defects in HNF1alpha trans-activation. In addition, our analysis sheds new light on the structure of a homeoprotein playing a key role in pancreatic beta cell function.

Multiple imprinted sense and antisense transcripts, differential methylation and tandem repeats in a putative imprinting control region upstream of mouse Igf2.

The mouse insulin-like growth factor 2 (Igf2) locus is a complex genomic region that produces multiple transcripts from alternative promoters. Expression at this locus is regulated by parental imprinting. However, despite the existence of putative imprinting control elements in the Igf2 upstream region, imprinted transcriptional repression is abolished by null mutations at the linked H19 locus. To clarify the extent to which the Igf2 upstream region contains autonomous imprinting control elements we have performed functional and comparative analyses of the region in the mouse and human. Here we report the existence of multiple, overlapping imprinted (maternally repressed) sense and antisense transcripts that are associated with a tandem repeat in the mouse Igf2 upstream region. Regions flanking the repeat exhibit tissue-specific parental allelic methylation patterns, suggesting the existence of tissue-specific control elements in the upstream region. Studies in H19 null mice indicate that both parental allelic methylation and monoallelic expression of the upstream transcripts depends on an intact H19 gene acting in cis. The homologous region in human IGF2 is structurally conserved, with the significant exception that it does not contain a tandem repeat. Our results support the proposal that tandem repeats act to target methylation to imprinted genetic loci.

Loss of the maternal H19 gene induces changes in Igf2 methylation in both cis and trans.

Recent investigations have shown that the maintenance of genomic imprinting of the murine insulin-like growth factor 2 (Igf2) gene involves at least two factors: the DNA (cytosine-5-)-methyltransferase activity, which is required to preserve the paternal specific expression of Igf2, and the H19 gene (lying 90 kb downstream of Igf2 gene), which upon inactivation leads to relaxation of the Igf2 imprint. It is not yet clear how these two factors are related to each other in the process of maintenance of Igf2 imprinting and, in particular, whether the latter is acting through cis elements or whether the H19 RNA itself is involved. By using Southern blots and the bisulfite genomic-sequencing technique, we have investigated the allelic methylation patterns (epigenotypes) of the Igf2 gene in two strains of mouse with distinct deletions of the H19 gene. The results show that maternal transmission of H19 gene deletions leads the maternal allele of Igf2 to adopt the epigenotype of the paternal allele and indicate that this phenomenon is influenced directly or indirectly by the H19 gene expression. More importantly, the bisulfite genomic-sequencing allowed us to show that the methylation pattern of the paternal allele of the Igf2 gene is affected in trans by deletions of the active maternal allele of the H19 gene. Selection during development for the appropriate expression of Igf2, dosage-dependent factors that bind to the Igf2 gene, or methylation transfer between the parental alleles could be involved in this trans effect.

The leptin receptor promoter controls expression of a second distinct protein.

The leptin receptor (OB-R) is a single membrane- spanning protein that mediates the weight-regulatory effects of leptin (OB protein). Several mRNA splice variants have been described which either encode OB-R proteins with cytoplasmic domains of different length or the OB-R and B219/OBR variants, which have different 5'-untranslated regions. Here we report evidence for the synthesis of a human mRNA splice variant of the OB-R gene that potentially encodes a novel protein, leptin receptor gene-related protein (OB-RGRP), which displays no sequence similarity to the leptin receptor itself. This OB-RGRP transcript contains the first two OB-R gene 5'-untranslated exons, but then is alternatively spliced to two novel exons which were mapped to a yeast artificial chromosome containing the leptin receptor gene. First identified by analysis of a large human expressed sequence tag database, the OB-RGRP transcript has now also been found in human and mouse tissues by the use of PCR. Preliminary experiments suggest that OB-RGRP and the OB-R variants share similar patterns of expression that are distinct from that of the B219/OBR variant. OB-RGRP is highly homologous to putative open reading frames in both yeast and Caenorhabditis elegans , suggesting a phylogenetically conserved role for this novel protein.

During in vivo maturation of eukaryotic nuclear mRNA, splicing yields excised exon circles.

Circular splicing has already been described on nuclear pre-mRNA for certain splice sites far apart in the multi exonic ETS-1 gene and in the single 1.2 kb exon of the Sry locus. To date, it is unclear how splice site juxtaposition occurs in normal and circular splicing. The splice site selection of an internal exon is likely to involve pairing between splice sites across that exon. Based on this, we predict that, albeit at low frequency, internal exons yield circular RNA by splicing as an error-prone mechanism of exon juxtaposition or, perhaps more interestingly, as a regulated mechanism on alternative exons. To address this question, the circular exon formation was analyzed at three ETS-1 internal exons (one alternative spliced exon and two constitutive), in human cell line and blood cell samples. Here, we show by RT-PCR and sequencing that exon circular splicing occurs at the three individual exons that we examined. RNase protection experiments suggest that there is no correlation between exon circle expression and exon skipping.

Regulatory elements in the first intron of the mouse Ha-ras gene.

The Ha-ras gene is one of the three oncogenes (Ha-ras, Ki-ras, and N-ras) of the ras superfamily of small G proteins. The p21ras proteins encoded by the ras genes are key proteins involved in the transduction of signals from membrane receptor-tyrosine kinases to downstream targets. The ras genes seem to play a ubiquitous role in the control of cell proliferation and cell differentiation. At the same time, ras genes may perform specific differentiated functions in certain cell types. Little is known about the regulation of expression of the Ha-ras gene. The first intron of the Ha-ras gene has been reported to be highly conserved between human and rodent. We investigated the role that this intron may play in the regulation of expression of Ha-ras. The promoter region of the Ha-ras gene exhibits characteristics of a housekeeping gene. Deletion analysis shows the existence of an enhancer-type element in the 5' region of the first intron (intron 0). DNase 1 footprinting experiments reveal five sites that interact with nuclear proteins from fibroblast and epithelial cell lines. Deletion and site-directed mutagenesis of three of these sites show that two are involved in a positive effect and one in a negative effect on the regulation of expression of the mouse Ha-ras gene.

An ATF/CREB-binding site is essential for cell-specific and inducible transcription of the murine MIP-1 beta cytokine gene.

The murine macrophage inflammatory protein 1 beta mRNA (MIP-1 beta) is rapidly and transiently induced in macrophages by lipopolysaccharide (LPS), serum or cycloheximide. Functional studies of the MIP-1 beta proximal promoter indicate that it is cell-specific, and serum- and LPS-responsive in macrophages. A 76-bp proximal promoter sequence (-51 to -127 bp) confers cell-specific and LPS-inducible activity when placed upstream from a heterologous promoter in both orientations. One essential cis-regulatory element within the enhancer-like sequence is an activating transcription factor/cAMP response element (CRE)-binding protein (ATF/CREB)-binding site, although the promoter is not cAMP responsive. Electrophoretic mobility shift assays and mutational analyses suggest that the promoter site is bound by nuclear protein complexes containing cAMP-independent members of the ATF/CREB family of proteins and c-Jun, and are functionally distinct from the AP1-related TPA-response element (TRE) binding activity.

Shift from fetal-type to Alzheimer-type phosphorylated Tau proteins in SKNSH-SY 5Y cells treated with okadaic acid.

Tau proteins are abnormally phosphorylated in Alzheimer's disease. Pathological Tau proteins named PHF-Tau 55, PHF-Tau 64, and PHF-Tau 69, are the main constituents of the paired helical filaments (PHF). When treating SKNSH-SY 5Y cells with okadaic acid (OA), Tau 55 protein was clearly induced whereas Tau 64 protein was only faintly induced. Here, we show that the absence of Tau 69 could be explained by the fact that adult isoforms containing N-terminal inserts are not detected. Phosphorylation is similar for untreated cellular Tau proteins and fetal Tau proteins, while OA cell treatment transformed fetal-type into Alzheimer-type phosphorylated proteins.

Mis-splicing yields circular RNA molecules.

We previously identified novel human ets-1 transcripts in which the normal order of exons is inverted, and demonstrated that although the order of exons is different than in the genomic DNA, splicing of these exons out of order occurs in pairs using genuine splice sites (1). Here we determine the structure of these novel transcripts, showing that they correspond to circular RNA molecules containing only exons in genomic order. These transcripts are stable molecules, localized in the cytoplasmic component of the cells. To our knowledge, this is the first case of circular transcripts being processed from nuclear pre-mRNA in eukaryotes. This new type of transcript might represent a novel aspect of gene expression and hold some interesting clues about the splicing mechanism.

Serum, AP-1 and Ets-1 stimulate the human ets-1 promoter.

The ets-1 proto-oncogene codes for a transcription factor. In order to understand how ets-1 is regulated, we have cloned its promoter. We show that the promoter is inducible by serum and expression of c-Fos and c-Jun, and it is positively auto-regulated by its gene product. A 50 base-pair sequence is sufficient to confer c-Fos + c-Jun and c-Ets-1 responsiveness to a heterologous promoter. This element contains two AP1 and one Ets-1 like motifs. Striking, AP-1 and Ets-1 motifs are found in oncogene responsive units (ORU's) of other promoters, suggesting that combining these motifs is a common mechanism for generating mitogen responsive transcription elements.

Mutation spectra of the two guanine adducts of the carcinogen 4-nitroquinoline 1-oxide in Escherichia coli. Influence of neighbouring base sequence on mutagenesis.

When the chemical carcinogen 4-nitroquinoline 1-oxide binds to DNA in vitro, two major adducts are formed, both at guanine residues, but at different positions, i.e. the C8 or the N2 position. Well-defined adducts (either C8 or N2 guanine adducts) can be formed in vitro by reacting DNA with 4-actoxyaminoquinoline 1-oxide (Ac-4HAQO) under different reaction conditions. Forward mutations induced by each of both main 4NQO adducts in the tetracycline resistance gene of pBR322 were determined. In total, 30 independent 4NQO-induced mutations were characterized, showing mainly base-pair substitution mutations and some frameshift mutations. We have observed that the 5' neighbouring base influences the specificity of dGuo-N2-AQO induced base-pair substitutions mutagenesis; a similar effect does not occur with dGuo-C8-AQO. This study reveals the importance of the N2 guanine adduct in the mutagenesis induced by 4NQO in vivo.

Splicing with inverted order of exons occurs proximal to large introns.

Following our studies which showed that the alpha and beta exons of the chicken c-ets-1 gene are not conserved in the human homologue, we succeeded in identifying a novel human c-ets-1 transcript in which the normal order of exons is scrambled. By PCR and RNase protection assays, we demonstrated that while the order of exons is different from that in genomic DNA, splicing of these exons in aberrant order occurs in pairs and at the same conserved consensus splice sites used in the normally spliced transcript. The scrambled transcript is non-polyadenylated and is expressed at much lower levels than the normal transcript. It is not the consequence of genomic rearrangement at the ets-1 locus nor is it due to the transcription of any ets-1 pseudogene. These results confirm previous observations of scrambled splicing.

Structural analysis of the mouse c-Ha-ras gene promoter.

Previous studies have demonstrated that the mouse c-Harvey ras proto-oncogene (c-Ha-ras) promoter sequences are GC rich and contain several potential transcription factor SP1 binding sites. We investigated the endonuclease hypersensitivity of this region in nuclei in vitro and whole mouse tissues in vivo and identified a very strong, ubiquitous hypersensitive site covering the proximal promoter sequences. Footprint protection studies using nuclear extracts from various cell types including fibroblasts, erythroid cells, and both normal and transformed epithelial cells revealed a consistent protein-binding pattern. Five protein binding sites were observed, four of which correlated with potential SP1 binding sites. Competition experiments using an oligonucleotide corresponding to a consensus SP1 binding site confirmed that these sequences were indeed bound by the SP1 (or SP1-like) trans-acting factor. In addition, no differences were observed between the footprint patterns obtained using extracts from cells of different lineages or between normal and transformed epithelial cells carrying activated ras genes. The controlling elements responsible for differential c-Ha-ras transcription between cell types or at different stages of carcinogenesis therefore probably lie in other regions of the gene.