Nutrigenomic profiling of reduced specification diets and phytogenic inclusion effects on critical toll-like receptor signaling, mitogen-activated protein kinase-apoptosis, and PI3K-Akt-mTOR gene components along the broiler gut.

The effects of concurrent reduction of dietary metabolizable energy (ME) and crude protein (CP) levels combined or not with the dietary inclusion of a phytogenic feed additive (PFA) were studied using a nutrigenomics approach. In particular, the expression of 26 critical genes relevant for inflammation control (TLR pathway), cellular apoptosis (MAPK pathway) cell growth and nutrient metabolism (PI3K-Akt-mTOR pathway) was profiled along the broiler intestine. Two dietary types (L and H) differing in metabolizable energy and crude protein levels (L: 95% and H: 100% of optimal Cobb 500 recommendations for ME and CP requirements) supplemented or not with PFA (- or +) and their interactions (L-, L+, H-, H+) were evaluated. There were only 3 total interactions (mTOR, IL8, and HRAS P < 0.05) between diet type and PFA inclusion indicating limited concurrent effects. Diet type, L upregulated genes related with inflammation mainly in the jejunum, ileum, and cecum (P < 0.05) and MAPK pathway in the ileum and cecum (P < 0.05). Moreover, diet type L negatively affected the expression of genes related to PI3K-Akt-mTOR pathway mainly in duodenum and cecum (P < 0.05). On the other hand, PFA inclusion downregulated (P < 0.05) genes related with TLR signaling pathway (TLR2B, MyD88, TLR3, IL8, LITAF) along the intestine and MAPK pathway genes (APO1, FOS) in jejunum (P < 0.05). Finally, PFA supplementation regulated nutrient sensing and metabolism in the cecum in a manner perceived as beneficial for growth. In conclusion, the study results highlight that the reduced ME and CP specifications, especially in the absence of PFA, regulate inflammation, apoptosis and nutrient metabolism processes at homeostatic control levels that hinder maximizing the availability of dietary energy and nutrients for growth purposes. Inclusion of PFA helped to adjust the respective homeostatic responses and control to levels supporting broiler performance, especially at reduced specification diets.

Characterization of the Dmrt1 gene in the black rockfish Sebastes schlegeli revealed a remarkable sex-dimorphic expression.

The Dmrt genes encode a large family of transcription factors with a conserved zinc finger-like DNA-binding DM domain. The function of Dmrt1, one of the family members, in sexual development has been well studied in invertebrates and vertebrates. In the present study, the full-length cDNA of Dmrt1 was isolated from the testis of Sebastes schlegeli. The full-length cDNA of S. schlegeli Dmrt1 (SsDmrt1) was 1,587 bp and contained a 189-bp 5' UTR, a 489-bp 3' UTR and a 909-bp open reading frame, which encoded 302 amino acids with a conserved DM domain and an male-specific motif domain. Phylogenetic analysis showed the evolutionary relationships of SsDmrt1 with other known Dmrt genes in fish and tetrapods. Several transcriptional factor-binding sites in the 5' promoter were identified that might regulate SsDmrt1 expression. Quantitative real-time PCR analysis indicated that SsDmrt1 was expressed in all of the inspected larval developmental stages from 1 to 35 days after birth and that the level of expression gradually decreased. The expression of SsDmrt1 in adult gonads was sexually dimorphic with extremely high expression in the testis, but very low expression in the ovary. No expression was detected in other tissues. Using in situ hybridization, we demonstrated that SsDmrt1 was specifically expressed in the germ cells of both the testis and the ovary. Thus, our results suggest that SsDmrt1 may have an important role in the differentiation of both the testis and the ovary of S. schlegeli.

An evolutionarily ancient NO synthase (NOS) in shrimp.

Nitric oxide (NO) is a well known essential molecule that is involved in multiple functions such as neuron transduction, cardiac disease, immune responses, etc.; nitric oxide synthase (NOS) is a critical enzyme that catalyzes the synthesis of it. A very few crustacean NOS molecules were biochemically characterized so far. In the present study, we cloned and characterized a NOS cDNA from haemocytes of tiger shrimp (Penaeus monodon) (PmNOS). The full-length of PmNOS cDNA contained 3997 bp, including a 5'UTR of 249 bp, ORF of 3582 bp and a 3'UTR of 166 bp. The putative peptide was 1193 amino acid residues in length, with an estimated molecular weight of 134.7 kDa and pI 6.7. Structurally, PmNOS contained oxygenase and reductase domains at N-terminal and C-terminal, respectively, and connected with a calmodulin binding motif. The deduced amino acid sequence of PmNOS shared 98% identical to the Chinese shrimp (Fenneropenaeus chinensis) NOS. Phylogenetically, PmNOS clustered with invertebrate NOS, but not clustered with iNOS, eNOS or nNOS found in vertebrates. PmNOS mRNA was expressed in many tissues or organs including thoracic and ventral nerves, midgut, gill, eyestalk, haemocytes, subcuticular epithelium and heart, but not found in hepatopancreas, muscle and lymphoid organ. But there was no significant difference in PmNOS mRNA expression after stimulation with LPS either by different concentration or time course or against CpG-ODN 2006. The enzyme activities of rPmNOS or crude homogenates from different tissues were detected, and were shown its highest activity in thoracic and ventral nerves, moderate in midgut and haemocytes but the lowest activity were seen in muscle. The addition of NOS antibody against NADPH binding domain leads to less activity which suggested that NADPH was an essential cofactor for PmNOS catalytic activity. The calcium dependency of PmNOS was ascertained using calmodulin inhibitor, Trifluroperazine. To confirm the population of haemocyte which produce NOS, the florescence test was assayed, and it implicated that the production of NO was catalyzed by subset of granulocytic NOS. Since the MW range, inducible/noninducible transcript, calcium-dependent activity and tissue distribution, we suggest that PmNOS may recognize as an ancient NOS evolutionarily.

The molecular evolution of the pif family proteins in various species of mollusks.

Various novel proteins have been identified from many kinds of mollusk shells. Although such matrix proteins are believed to play important roles in the calcium carbonate crystal formation of shells, no common proteins that interact with calcium carbonate or that are involved in the molecular mechanisms behind shell formation have been identified. Pif consists of two proteins, Pif 80 and Pif 97, which are encoded by a single mRNA. Pif 80 was identified as a key acidic protein that regulates the formation of the nacreous layer in Pinctada fucata, while Pif 97 has von Willebrand factor type A (VWA) and chitin-binding domains. In this study, we identified Pif homologues from Pinctada margaritifera, Pinctada maxima, Pteria penguin, Mytilus galloprovincialis, and in the genome database of Lottia gigantea in order to compare their primary protein sequences. The VWA and chitin-binding domains are conserved in all Pif 97 homologues, whereas the amino acid sequences of the Pif 80 regions differ markedly among the species. Sequence alignment revealed the presence of a novel significantly conserved sequence between the chitin-binding domain and the C-terminus of Pif 97. Further examination of the Pif 80 regions suggested that they share a sequence that is similar to the laminin G domain. These results indicate that all Pif molecules in bivalves and gastropods may be derived from a common ancestral gene. These comparisons may shed light on the correlation between molecular evolution and morphology in mollusk shell microstructure.

Cloning and characterization of boron transporters in Brassica napus.

Six full-length cDNA encoding boron transporters (BOR) were isolated from Brassica napus (AACC) by rapid amplification of cDNA ends (RACE). The phylogenic analysis revealed that the six BORs were the orthologues of AtBOR1, which formed companying with the triplication and allotetra-ploidization process of B. napus, and were divided into three groups in B. napus. Each group was comprised of two members, one of which was originated from Brassica rapa (AA) and the other from Brassica oleracea (CC). Based on the phylogenetic relationships, the six genes were named as BnBOR1;1a, BnBOR1;1c, BnBOR1;2a, BnBOR1;2c, BnBOR1;3a and BnBOR1;3c, respectively. The deduced BnBOR1 s had extensive similarity with other plant BORs, with the identity of 74-96.8% in amino acid sequence. The BnBOR1;3a and BnBOR1;3c resembled AtBOR1 in number and positions of the 11 introns, but the others only have 9 introns. After the gene duplication, there was evidence of purifying selection under a divergent selective pressure. The expression patterns of the six BnBOR1 s were detected by semi-quantitative RT-PCR. The BnBOR1;3a and BnBOR1;3c showed a ubiquitous expression in all of the investigated tissues, whereas the other four genes showed similar tissue-specific expression profile. Unlike the non-transcriptional regulation of AtBOR1, the expression of BnBOR1;1c and BnBOR1;2a were obviously induced by boron deficiency. This study suggested that the BOR1 s had undergone a divergent expression pattern in the genome of B. napus after that the B. napus diverged from Arabidopsis thaliana.

Genomic characterization, phylogeny and gene regulation of g-type lysozyme in sole (Solea senegalensis).

The g-type lysozyme is a key protein of the innate immune system to fight bacterial infections. In this study we cloned and characterized the gene encoding for g-type lysozyme in Senegalese sole (Solea senegalensis). The deduced amino acid sequence comprised 195 residues containing the three conserved catalytic residues and two cysteines. A BAC analysis revealed that the gene is structured in 5 exons and 4 introns. Also, two polyadenylation signals that generate two cDNAs differing in 3'-UTR length were detected. Promoter analysis showed the presence of the main cis-acting elements involved in the transcriptional regulation of the gene. At genomic level, the g-type lysozyme was associated with mucolipin 1 and the peptidoglycan recognition protein 2 conforming a cluster of antidefensive genes with a well-conserved synteny across Percomorpha. FISH analysis using the BAC clone revealed a single hybridization signal located in an acrocentric chromosome pair. The phylogenetic analysis confirmed that the g-type lysozyme represents a complex group in fish that has been shaped by gene duplications and diversification with several positions under Darwinian selection. Expression analysis in juvenile tissues indicated that transcript levels were higher in gills, spleen and heart. During development, gene expression activated just at the beginning of metamorphosis, increasing progressively until climax. Hormonal treatments demonstrated that this gene was regulated positively by thyroid hormones during development and negatively by dexamethasone. In contrast, no response was observed after all-trans retinoic acid or 4-diethylaminobenzaldehyde treatments. Finally, treatments using lipopolysaccharide, lipoteichoic acid, peptidoglycan, zymosan and poly(I:C) activated gene expression in a time- and tissue-specific manner. Taken together, data indicate that g-type lysozyme is a high evolutionary conserved gene that diversified to adapt to changing environment and pathogen conditions. Gene expression can be activated by diverse pathogen stimuli and modulated by physiological factors with important consequences for the aquaculture of this species.

Identification and cloning of a transglutaminase from giant freshwater prawn, Macrobrachium rosenbergii, and its transcription during pathogen infection and moulting.

Complementary (c)DNA encoding transglutaminase (TG) messenger (m)RNA of the giant freshwater prawn, Macrobrachium rosenbergii, was cloned from haemocytes by a reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) using oligonucleotide primers based on the TG sequence of the horseshoe crab, Tachypleus tridentatus; tiger shrimp, Penaeus monodon; kuruma shrimp, Marsupenaeus japonicus; and crayfish, Pacifastacus leniusculus. The 2722-bp cDNA contained an open reading frame (ORF) of 2334 bp, a 72-bp 5'-untranslated region (UTR), and a 316-bp 3'-UTR containing a stop codon and a poly A tail. The molecular mass of the deduced amino acid (aa) sequence (778 aa) was 86.67 kDa with an estimated pI of 5.4. The M. rosenbergii TG (abbreviated MrTG, accession no.: JF309296) contains a typical transglutaminase-like homologue, two putative integrin-binding motifs (RGD), ten glycosylation sites, and four calcium-binding sites; a catalytic triad is present as in arthropod TGs. Sequence comparison and a phylogenetic analysis revealed that shrimp TG can be separated into three subgroups, penaeid TG1, freshwater crustacean TG2 and marine crustacean TG2, and MrTG was more closely related to TG2 than to TG1. MrTG mRNA and TG activities were detected in all tested tissues of M. rosenbergii, with MrTG mainly being synthesised by haemocytes. There was a negative correlation between clotting time of haemolymph, and MrTG expression and TG activity of haemocytes in prawn injected with Lactococcus garvieae. The pattern of MrTG mRNA expression and TG activity in haemocytes exhibited a contrary tendency with clotting time of haemolymph during the moult stages. Those results indicate that cloned MrTG is involved in the defence response, and is probably the major functional TG for haemolymph coagulation in M. rosenbergii.

Gene structure analysis of kisspeptin-2 (Kiss2) in the Senegalese sole (Solea senegalensis): characterization of two splice variants of Kiss2, and novel evidence for metabolic regulation of kisspeptin signaling in non-mammalian species.

Kisspeptin signaling in the brain is involved in the control of the onset of puberty in vertebrates. In this study, we present novel evidence indicating that kisspeptin may link energy balance and reproduction. For that purpose, we determined the complete gene structure of kisspeptin in a teleost fish, the Senegalese sole (Ss). In contrast to the situation evident in several fish, in this species only Kiss2 was found. Yet, two Ss Kiss2 isoforms generated by alternative splicing through intronic retention were detected: Ss Kiss2_v1, producing the functional protein, and Ss Kiss2_v2, coding for a truncated, non-functional protein. Specific qPCRs showed that the expression of these two isoforms varied differently in brain and gonads throughout maturation. In addition, and in contrast to what has been observed in mammals, fasting increased hypothalamic mRNA levels of Ss Kiss2_v1, which also caused a concomitant rise in pituitary Ss LH and Ss FSH mRNA. Together, these data indicate the impact of the nutritional status on Kiss mRNA expression as a potential regulatory mechanism for the metabolic control of reproduction in non-mammalian species, albeit with some significant differences with respect to the situation described in mammals.

Evolution of nematode-resistant Mi-1 gene homologs in three species of Solanum.

Plants have evolved several defense mechanisms, including resistance genes. Resistance to the root-knot nematode Meloidogyne incognita has been found in wild plant species. The molecular basis for this resistance has been best studied in the wild tomato Solanum peruvianum and it is based on a single dominant gene, Mi-1.2, which is found in a cluster of seven genes. This nematode attacks fiercely several crops, including potatoes. The genomic arrangement, number of copies, function and evolution of Mi-1 homologs in potatoes remain unknown. In this study, we analyzed partial genome sequences of the cultivated potato species S. tuberosum and S. phureja and identified 59 Mi-1 homologs. Mi-1 homologs in S. tuberosum seem to be arranged in clusters and located on chromosome 6 of the potato genome. Previous studies have suggested that Mi-1 genes in tomato evolved rapidly by frequent sequence exchanges among gene copies within the same cluster, losing orthologous relationships. In contrast, Mi-1 homologs from cultivated potato species (S. tuberosum and S. phureja) seem to have evolved by a birth-and-death process, in which genes evolve mostly by mutations and interallelic recombinations in addition to sequence exchanges.

Molecular and functional analyses of aspolin, a fish-specific protein extremely rich in aspartic acid.

Aspolin is a muscular protein having unique structural characteristics where the most part of its primary structure is occupied by aspartic acid. Aspolin has been found exceptionally in fish muscle, suggesting its specific role in this tissue. However, biological functions of aspolin have remained unknown. In the present study, we cloned full-length cDNAs encoding zebrafish Danio rerio aspolins 1 and 2, revealed their genomic organization, and examined in vivo function using knockdown techniques. Genomic analysis clearly showed that aspolin is a paralog of the histidine-rich calcium binding protein gene, which encodes a calcium binding protein in sarcoplasmic reticulum (SR). Expression analysis showed that the transcripts and their translated products, aspolins 1 and 2, are distributed in myotomal skeletal muscle, but not in cardiac muscle. Injection of antisense morpholino oligo targeting both aspolins 1 and 2 increased the mRNA levels of calsequestrin 1, another calcium binding protein in SR. These lines of evidence suggest that aspolins regulate calcium concentrations in SR.

Cloning, characterization, expression, and copper sensitivity of the metallothionein-1 gene in the Chinese mitten crab, Eriocheir sinensis.

A full-length metallothionein-1(MT-1) cDNA was cloned from the Chinese mitten crab, Eriocheir sinensis, based upon the hepatopancreas cDNA library. The full-length cDNA contained a single 180 bp open reading frame that encoded a 59 amino acid protein. The deduced amino acid sequence was cysteine (Cys)-rich, with residues observed in patterns characteristic of other reported MTs: Cys-X-Cys, Cys-X-X-Cys, or Cys-X-X-X-Cys. Gene structure obtained via PCR yielded a 3816 bp gene, which was comprised of three exons and two introns arranged in a "3 + 2" pattern. The cloned 5'flanking region (1,735 bp) contained several predicted binding sites, which included MREs, AP-1, SP1, USF, GATA, HNF-1, and HSF. MT-1 mRNA expression analysis revealed that while levels were highest in the hepatopancreas, expression was abundant in testis and thoracic ganglia, moderate in intestine (P<0.05), and weak in other tissues (P<0.05). MT-1 mRNA expression exhibited reproductive variation in the male, with levels approximately tenfold greater in August, during seasonal gonadal maturation, compared to other times of the year. Cu2+ exposure via tank water (0-1 mg/l for 7 days) resulted in a dose-dependent bell curve response in MT-1 mRNA expression, with peak expression observed after exposure to 0.1 mg/l Cu2+. A time course experiment (0.1 mg/l Cu2+ over 9 days) revealed MT-1 mRNA expression peaked sharply on day 5 before gradually decreasing with prolonged exposure. In the present report, we provide sequence analysis of the first MT-1 gene cloned in E. sinensis, and evidence that its physiological and toxicological regulation is evolutionary conserved.

Rice pollen hybrid incompatibility caused by reciprocal gene loss of duplicated genes.

Genetic incompatibility is a barrier contributing to species isolation and is caused by genetic interactions. We made a whole genome survey of two-way interacting loci acting within the gametophyte or zygote using independence tests of marker segregations in an F(2) population from an intersubspecific cross between O. sativa subspecies indica and japonica. We detected only one reproducible interaction, and identified paralogous hybrid incompatibility genes, DOPPELGANGER1 (DPL1) and DOPPELGANGER2 (DPL2), by positional cloning. Independent disruptions of DPL1 and DPL2 occurred in indica and japonica, respectively. DPLs encode highly conserved, plant-specific small proteins (∼10 kDa) and are highly expressed in mature anther. Pollen carrying two defective DPL alleles became nonfunctional and did not germinate, suggesting an essential role for DPLs in pollen germination. Although rice has many duplicated genes resulting from ancient whole genome duplication, the origin of this gene duplication was in recent small-scale gene duplication, occurring after Oryza-Brachypodium differentiation. Comparative analyses suggested the geographic and phylogenetic distribution of these two defective alleles, showing that loss-of-function mutations of DPL1 genes emerged multiple times in indica and its wild ancestor, O. rufipogon, and that the DPL2 gene defect is specific to japonica cultivars.

Recombinant yeast screen for new inhibitors of human acetyl-CoA carboxylase 2 identifies potential drugs to treat obesity.

Acetyl-CoA carboxylase (ACC) is a key enzyme of fatty acid metabolism with multiple isozymes often expressed in different eukaryotic cellular compartments. ACC-made malonyl-CoA serves as a precursor for fatty acids; it also regulates fatty acid oxidation and feeding behavior in animals. ACC provides an important target for new drugs to treat human diseases. We have developed an inexpensive nonradioactive high-throughput screening system to identify new ACC inhibitors. The screen uses yeast gene-replacement strains depending for growth on cloned human ACC1 and ACC2. In "proof of concept" experiments, growth of such strains was inhibited by compounds known to target human ACCs. The screen is sensitive and robust. Medium-size chemical libraries yielded new specific inhibitors of human ACC2. The target of the best of these inhibitors was confirmed with in vitro enzymatic assays. This compound is a new drug chemotype inhibiting human ACC2 with 2.8 muM IC(50) and having no effect on human ACC1 at 100 muM.

Aspergillus fumigatus survival in alkaline and extreme zinc-limiting environments relies on the induction of a zinc homeostasis system encoded by the zrfC and aspf2 genes.

Aspergillus fumigatus has three zinc transporter-encoding genes whose expression is regulated by both pH and the environmental concentration of zinc. We have previously reported that the zrfA and zrfB genes of A. fumigatus are transcribed at higher levels and are required for fungal growth under acidic zinc-limiting conditions whereas they are dispensable for growth in neutral or alkaline zinc-limiting media. Here we report that the transporter of the zinc uptake system that functions in A. fumigatus growing in neutral or alkaline environments is encoded by zrfC. The transcription of zrfC occurs divergently with respect to the adjacent aspf2 gene, which encodes an immunodominant antigen secreted by A. fumigatus. The two genes-zrfC and aspf2-are required to different extents for fungal growth in alkaline and extreme zinc-limiting media. Indeed, these environmental conditions induce the simultaneous transcription of both genes mediated by the transcriptional regulators ZafA and PacC. ZafA upregulates the expression of zrfC and aspf2 under zinc-limiting conditions regardless of the ambient pH, whereas PacC represses the expression of these genes under acidic growth conditions. Interestingly, the mode of action of PacC for zrfC-aspf2 transcription contrasts with the more widely accepted model for PacC function, according to which under alkaline growth conditions PacC would activate the transcription of alkaline-expressed genes but would repress the transcription of acid-expressed genes. In sum, this report provides a good framework for investigating several important aspects of the biology of species of Aspergillus, including the repression of alkaline genes by PacC at acidic pH and the interrelationship that must exist between tissue pH, metal availability in the host tissue, and fungal virulence.

Differential analysis for high density tiling microarray data.

BACKGROUND: High density oligonucleotide tiling arrays are an effective and powerful platform for conducting unbiased genome-wide studies. The ab initio probe selection method employed in tiling arrays is unbiased, and thus ensures consistent sampling across coding and non-coding regions of the genome. These arrays are being increasingly used to study the associated processes of transcription, transcription factor binding, chromatin structure and their association. Studies of differential expression and/or regulation provide critical insight into the mechanics of transcription and regulation that occurs during the developmental program of a cell. The time-course experiment, which comprises an in-vivo system and the proposed analyses, is used to determine if annotated and un-annotated portions of genome manifest coordinated differential response to the induced developmental program. RESULTS: We have proposed a novel approach, based on a piece-wise function - to analyze genome-wide differential response. This enables segmentation of the response based on protein-coding and non-coding regions; for genes the methodology also partitions differential response with a 5' versus 3' versus intra-genic bias. CONCLUSION: The algorithm built upon the framework of Significance Analysis of Microarrays, uses a generalized logic to define regions/patterns of coordinated differential change. By not adhering to the gene-centric paradigm, discordant differential expression patterns between exons and introns have been identified at a FDR of less than 12 percent. A co-localization of differential binding between RNA Polymerase II and tetra-acetylated histone has been quantified at a p-value < 0.003; it is most significant at the 5' end of genes, at a p-value < 10-13. The prototype R code has been made available as supplementary material [see Additional file 1].

Calcium-activated potassium channel of the tobacco hornworm, Manduca sexta: molecular characterization and expression analysis.

Large-conductance calcium- and voltage-gated potassium channels (BK or Slowpoke) serve as dynamic integrators linking electrical signaling and intracellular activity. These channels can mediate many different Ca2+-dependent physiological processes including the regulation of neuronal and neuroendocrine cell excitability and muscle contraction. To gain insights into the function of BK channels in vivo, we isolated a full-length cDNA encoding the alpha subunit of a Slowpoke channel from the tobacco hornworm, Manduca sexta (msslo). Amino acid sequence comparison of the deduced Manduca protein revealed at least 80% identity to the insect Slo channels. The five C-terminal alternative splice regions are conserved, but the cloned cDNA fragments contained some unique combinations of exons E, G and I. Our spatial profile revealed that transcript levels were highest in skeletal muscle when compared with the central nervous system (CNS) and visceral muscle. The temporal profile suggested that msslo expression is regulated developmentally in a tissue- and regional-specific pattern. The levels of msslo transcripts remain relatively constant throughout metamorphosis in the CNS, transiently decline in the heart and are barely detectable in the gut except in adults. A dramatic upregulation of msslo transcript levels occurs in thoracic but not abdominal dorsal longitudinal body wall muscles (DLM), suggesting that the msSlo current plays an important role in the excitation or contractile properties of the phasic flight muscle. Our developmental profile of msslo expression suggests that msSlo currents may contribute to the changes in neural circuits and muscle properties that produce stage-specific functions and behaviors.

Identification and characterization of novel peroxisome proliferator-activated receptor-gamma (PPAR-gamma) transcriptional variants in pig and human.

The peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is a member of the steroid/thyroid/retinoid receptor superfamily, and is primarily expressed in fat tissue. To date, two major PPAR-gamma isoforms have been identified in pig, PPAR-gamma1 and PPAR-gamma2. Porcine PPAR-gamma1a consists of two leader exons, designated A1 and A2, followed by six exons containing the open reading frame. Here, we report the isolation and characterization of three novel PPAR-gamma1 transcripts. PPAR-gamma1b is derived from exon A1, with exon A2 spliced out. PPAR-gamma1c and PPAR-gamma1d are derived from the new exon, A', containing exon A2 (gamma1c) or without exon A2 (gamma1d). Based on PCR analysis of PAC clones that included sequences from the 5'-untranslated region of the PPAR-gamma gene, the new A' exon is located between the known exons A1 and A2. We also isolated the human homologue to exon A', as well as the two new PPAR-gamma1c and -gamma1d splice variants, from human adipose tissue. Studies of the expression of porcine PPAR-gamma by real time reverse transcription-polymerase chain reaction analysis show that transcripts derived from exon A1 were not expressed at significantly different levels in visceral fat (lamina subserosa) or subcutaneous fat (back fat, inner and outer layer). In contrast, exon A'-derived transcripts were expressed at progressively higher levels in the inner and outer layers of subcutaneous fat than in visceral fat. The same expression pattern was also observed for PPAR-gamma2. We hypothesize that there are three promoters, which differentially regulate PPAR-gamma1 and PPAR-gamma2 gene expression, depending on the specific localization of the fat tissue.

Ancient DNA from pollen: a genetic record of population history in Scots pine.

Assessments of plant population dynamics in space and time have depended on dated records of fossil pollen synthesized on a subcontinental scale. Genetic analyses of extant populations have revealed spatial relationships that are indicative of past spatial dynamics, but lack an explicit timescale. Synthesis of these data requires genetic analyses from abundant dated fossil material, and this has hitherto been lacking. Fossil pollen is the most abundant material with which to fill this data gap. Here we report genetic analyses of fossil pollen retrieved from Holtjärnen postglacial lake sediment in Sweden and show that plastid DNA is recoverable from Scots Pine and Norway spruce pollen grains that are 100 and 10 000 years old. By sequencing clones from two short plastid PCR products and by using multiple controls we show that the ancient sequences were endogenous to the fossil grains. Comparison of ancient sequences and those obtained from an extant population of Scots pine establishes the first genetic link between extant and fossil samples in this species, providing genetic continuity through time. The finding of one common haplotype present in modern, 100-year old and 10 000-year old samples suggests that it may have persisted near Holtjärnen throughout the postglacial period. This retrieval of ancient DNA from pollen has major implications for plant palaeoecology in conifer species by allowing direct estimates of population dynamics in space and time.

N-acylphosphatidylethanolamine-hydrolyzing phospholipase D: a novel enzyme of the beta-lactamase fold family releasing anandamide and other N-acylethanolamines.

N-acylethanolamines (NAEs) are a lipid class present in brain and other animal tissues and contains anandamide (an endocannabinoid) and other bioactive substances. NAEs are formed from N-acylphosphatidylethanolamines (NAPEs) by a phospholipase D (PLD)-type enzyme abbreviated to NAPE-PLD. Although this enzyme has been recognized for more than 20 years, its molecular cloning has only recently been achieved by us. We highly purified NAPE-PLD from the particulate fraction of rat heart, and on the basis of peptide sequences with the purified enzyme cloned its cDNA from mouse, rat and human. The deduced primary structures revealed no homology with any PLDs so far reported, but was suggested to belong to the beta-lactamase fold family. When overexpressed in COS-7 cells, the NAPE-PLD activity increased about 1000-fold in comparison with the endogenous activity. The recombinant enzyme generated various long-chain NAEs including anandamide from their corresponding NAPEs at similar rates. However, the enzyme was inactive with phosphatidylethanolamine and phosphatidylcholine and did not catalyze transphosphatidylation, a reaction characteristic of PLD. The enzyme was widely expressed in murine organs with higher levels in brain, testis and kidney. The existence of NAPE-PLD specifically hydrolyzing NAPEs to NAEs emphasizes physiological significance of NAEs including anandamide in brain and other tissues.

Highly unsaturated fatty acid synthesis in vertebrates: new insights with the cloning and characterization of a delta6 desaturase of Atlantic salmon.

Fish are an important source of the n-3 highly unsaturated fatty acids (HUFA), eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids that are crucial to the health of higher vertebrates. The synthesis of HUFA involves enzyme-mediated desaturation, and a delta5 fatty acyl desaturase cDNA has been cloned from Atlantic salmon (Salmo salar) and functionally characterized previously. Here we report cloning and functional characterization of a delta6 fatty acyl desaturase of Atlantic salmon and describe its genomic structure, tissue expression, and nutritional regulation. A salmon genomic library was screened with a salmon delta5 desaturase cDNA and positive recombinant phage isolated and subcloned. The full-length cDNA for the putative fatty acyl desaturase was shown to comprise 2106 bp containing an open reading frame of 1365 bp specifying a protein of 454 amino acids (GenBank accession no. AY458652). The protein sequence included three histidine boxes, two transmembrane regions, and an N-terminal cytochrome b5 domain containing the heme-binding motif HPGG, all of which are characteristic of microsomal fatty acid desaturases. Functional expression showed that this gene possessed predominantly delta6 desaturase activity. Screening and sequence analysis of the genomic DNA of a single fish revealed that the delta6 desaturase gene constituted 13 exons in 7965 bp of genomic DNA. Quantitative real-time PCR assay of gene expression in Atlantic salmon showed that both delta6 and delta5 fatty acyl desaturase genes, and a fatty acyl elongase gene, were highly expressed in intestine, liver, and brain, and less so in kidney, heart, gill, adipose tissue, muscle, and spleen. Furthermore, expression of both delta6 and delta5 fatty acyl desaturase genes in intestine, liver, red muscle, and adipose tissue was higher in salmon fed a diet containing vegetable oil than in fish fed a diet containing fish oil.