Identifying protein-coding genes in genomic sequences.

The vast majority of the biology of a newly sequenced genome is inferred from the set of encoded proteins. Predicting this set is therefore invariably the first step after the completion of the genome DNA sequence. Here we review the main computational pipelines used to generate the human reference protein-coding gene sets.

New anthropometry-based age- and sex-specific reference values for urinary 24-hour creatinine excretion based on the adult Swiss population (epublish)

BACKGROUND: Urinary creatinine excretion is used as a marker of completeness of timed urine collections, which are a keystone of several metabolic evaluations in clinical investigations and epidemiological surveys. METHODS: We used data from two independent Swiss cross-sectional population-based studies with standardised 24-hour urinary collection and measured anthropometric variables. Only data from adults of European descent, with estimated glomerular filtration rate (eGFR) ≥60 ml/min/1.73 m2 and reported completeness of the urinary collection were retained. A linear regression model was developed to predict centiles of the 24-hour urinary creatinine excretion in 1,137 participants from the Swiss Survey on Salt and validated in 994 participants from the Swiss Kidney Project on Genes in Hypertension. RESULTS: The mean urinary creatinine excretion was 193 ± 41 μmol/kg/24 hours in men and 151 ± 38 μmol/kg/24 hours in women in the Swiss Survey on Salt. The values were inversely correlated with age and body mass index (BMI). CONCLUSIONS: We propose a validated prediction equation for 24-hour urinary creatinine excretion in the general European population, based on readily available variables such as age, sex and BMI, and a few derived normograms to ease its clinical application. This should help healthcare providers to interpret the completeness of a 24-hour urine collection in daily clinical practice and in epidemiological population studies.

Expression of apoptosis and survival genes in human memory T cell populations

RESUME Introduction: Les cellules T mémoires humaines sont classées en trois sous-populations sur la base de l'expression d'un marqueur de surface cellulaire, CD45RA, et du récepteur aux chimiokines, CCR7. Ces sous-populations, nommées cellules mémoires centrales (TcM), mémoires effectrices (TEM) et mémoires effectrices terminales (ITEM), ont des rôles fonctionnels distincts, ainsi que des capacités de prolifération et de régénération différentes. Cependant, la génération de ces différences reste encore mal comprise et on ignore les mécanismes moléculaires impliqués. Matériaux et Méthodes: Des cellules mononucléaires humaines du sang périphérique ont été séparées par cytométrie de flux selon leur expression de CD4, CD8, CD45RA et CCR7 en sous-populations de cellules CD4+ ou CD8+ naïves, TcM, TEM ou ITEM. Dans chacune de ces sous-populations, 14 gènes impliqués dans l'apoptose, la survie ou la capacité proliférative des cellules T ont été quantifiés par RT-PCR en temps réel, relativement à l'expression d'un gène de référence endogène. L'ARN provenant de 450 cellules T a été utilisé par gène et par sous-population. Les gènes analysés (cibles) comprenaient des gènes de survie (BAFF, APRIL, BAFF-R, BCMA, TACI, IL-15Rα, IL-7Rα), des gènes anti-apoptotiques (Bcl-2, BclxL, FLIP), des gènes pro-apoptotiques (Bad, Bax, Fast) et le gène anti-prolifératif, Tob. A l'aide de la méthode comparative delta-delta-CT, le taux d'expression des gènes cibles de chaque sous-population des cellules T mémoires CD4+ et CD8+, à été comparée à leur taux d'expression dans les cellules T naïves CD4+ et CD8+. Résultats: Dans les cellules CD8+, les gènes pro-apoptotiques Bax et Fast étaient surexprimés dans toutes les sous-populations mémoires, tandis que l'expression des facteurs anti-apoptotiques et de survie comme Bcl-2, APRIL et BAFF-R, étaient diminués. Ces deux tendances étaient particulièrement accentuées dans les sous-groupes des cellules mémoires TEM et TTEM. A noter que malgré le fait que leur expression était également diminuée dans les autres cellules mémoires, le facteur de survie IL-7Ra, était sélectivement surexprimé dans la sous-population de cellules TcM et l'expression d'IL-15Ra était sélectivement augmentée dans les TEM. Dans les cellules CD4+, le taux d'expression des gènes analysés était plus variable entre les sujets étudiés que dans les cellules CD8+, ne permettant pas de définir un profil d'expression spécifique. L'expression du gène de survie BAFF par contre, a été significativement augmentée dans toutes les sous-populations mémoire CD4+. Il en va de même pour l'expression d' APRIL et de BAFF-R, bien que dans moindre degré. A remarquer que l'expression du facteur anti-apoptotique Fast a été observée uniquement dans la souspopulation des TTEM. Discussion et Conclusions: Cette étude montre une nette différence entre les cellules CD8+ et CD4+, en ce qui concerne les profils d'expression des gènes impliqués dans la survie et l'apoptose des cellules T mémoires. Ceci pourrait impliquer une régulation cellulaire homéostatique distincte dans ces deux compartiments de cellules T mémoires. Dans les cellules CD8+ l'expression d'un nombre de gènes impliqués dans la survie et la protection de l'apoptose semblerait être diminuée dans les populations TEM et TTEM en comparaison à celle des sous-populations naïves et TEM, tandis que l'expression des gènes pro-apoptotiques semblerait être augmentée. Comme ceci paraît être plus accentué dans les TTEM, cela pourrait indiquer une plus grande disposition à l'apopotose dans les populations CCR7- (effectrices) et une perte de survie parallèlement à l'acquisition de capacités effectrices. Ceci parlerait en faveur d'un modèle de différentiation linéaire dans les cellules CD8+. De plus, l'augmentation sélective de l'expression d'IL-7Ra observée dans le sous-groupe de cellules mémoires TEM, et d'IL-15Ra dans celui des TEM, pourrait indiquer un moyen de sélection pour des réponses immunitaires mémoires à long terme par une réponse distincte à ces cytokines. Dans les cellules CD4+ par contre, aucun profil d'expression n'a pu être déterminé; les résultats suggèrent même une résistance relative à l'apoptose de la part des cellules mémoires. Ceci pourrait favoriser l'existence d'un modèle de différentiation plus flexible avec des possibilités d'interaction multiples. Ainsi, la surexpression sélective de BAFF, APRIL et BAFF-R dans les sous-populations individuelles des cellules mémoires pourrait être un indice de l'interaction de ces sous-groupes avec des cellules B. ABSTRACT Introduction: Based on their surface expression of the CD45 isoform and of the CCR7 chemokine receptor, memory T cells have been divided into the following three subsets: central memory (TAM), effector memory (TEM) and terminal effector memory (ITEM). Distinct functional roles and different proliferative and regenerative capacities have been attributed to each one of these subpopulations. The molecular mechanisms underlying these differences; however, remain poorly understood. Materials and Methods: According to their expression of CD4, CD8, CD45RA and CCR7, human peripheral blood mononuclear cells were sorted by flow-cytometry into CD4+ or CD8+ naïve, TAM, TEM and ITEM subsets. Using real-time PCR, the expression of 14 genes known to be involved in apoptotis, survival or proliferation of T cells was quantified separately in each individual subset, relative to an endogenous reference gene. The RNA equivalent of 450 T cells was used for each gene and subset. The target gene panel included the survival genes BAFF, APRIL, BAFF-R, BCMA, TACI, IL-15Rα and IL-7Rα, the anti-apoptotic genes Bcl2, Bcl-xL and FLIP, the pro-apoptotic genes Bad, Bax and Fast, as well as the antiproliferative gene Tob. Using the comparative CT-method, the expression of the target genes in the three memory T cell subsets of both CD4+ and CD8+ T cell populations was compared to their expression in the naïve T cells. Results: In CD8+ cells, the pro-apoptotic factors Bax and Fast were found to be upregulated in all memory T cell subsets, whereas the survival and anti-apoptotic factors Bcl-2, APRIL and BAFF-R were downregulated. These tendencies were most accentuated in TEM and TTEM subsets. Even though the survival factor IL-7Rα was also downregulated in these subsets, interestingly, it was selectively upregulated in the CD8+ TAM subset. Similarly, IL-15Rαexpression was shown to be selectively upregulated in the CD8+ TEM subset. In CD4+ cells, the expression levels of the analyzed genes showed a greater inter-individual variability than in CD8+ cells, thus suggesting the absence of any particular expression pattern for CD4+ memory T cells. However, the survival factor BAFF was found to be significantly upregulated in all CD4+ memory T cell subsets, as was also the expression of APRIL and BAFF-R, although to a lesser extent. Furthermore, it was noted that the pro-apoptotic gene Fast was only expressed in the TTEM CD4+ subset. Discussion and Conclusions: Genes involved in apoptosis and survival in human memory T cells have been shown to be expressed differently in CD8+ cells as compared to CD4+ cells, suggesting a distinct regulation of cell homeostasis in these two memory T cell compartments. The present study suggests that, in CD8+ T cells, the expression of various survival and antiapoptotic genes is downregulated in TEM and TTEM subsets, while the expression of proapoptotic genes is upregulated in comparison to the naïve and the TAM populations. These characteristics, potentially translating to a greater susceptibility to apoptosis in the CCR7- (effector) memory populations, are accentuated in the TTEM population, suggesting a loss of survival in parallel to the acquisition of effector capacities. This speaks in favour of a linear differentiation model in CD8+ T memory cells. Moreover, the observed selectively increased expression of IL-7Rα in CD8+ TAM cells - as that of IL-15Rα in CD8+ TEM cells -suggest that differential responsiveness to cytokines could confer a selection bias for distinct long-term memory cell responses. Relative to the results for CD8+ T cells, those for CD4+ T cells seem to indicate a certain resistance of the memory subsets to apoptosis, suggesting the possibility of a more flexible differentiation model with multiple checkpoints and potential interaction of CD4+ memory cells with other cells. Thus, the selective upregulation of BAFF, APRIL and BAFF-R in individual memory subsets could imply an interaction of these subsets with B cells.

Systems Genetics of Metabolism: The Use of the BXD Murine Reference Panel for Multiscalar Integration of Traits.

Metabolic homeostasis is achieved by complex molecular and cellular networks that differ significantly among individuals and are difficult to model with genetically engineered lines of mice optimized to study single gene function. Here, we systematically acquired metabolic phenotypes by using the EUMODIC EMPReSS protocols across a large panel of isogenic but diverse strains of mice (BXD type) to study the genetic control of metabolism. We generated and analyzed 140 classical phenotypes and deposited these in an open-access web service for systems genetics (www.genenetwork.org). Heritability, influence of sex, and genetic modifiers of traits were examined singly and jointly by using quantitative-trait locus (QTL) and expression QTL-mapping methods. Traits and networks were linked to loci encompassing both known variants and novel candidate genes, including alkaline phosphatase (ALPL), here linked to hypophosphatasia. The assembled and curated phenotypes provide key resources and exemplars that can be used to dissect complex metabolic traits and disorders.

Reference values and factors associated with renal resistive index in a family-based population study.

Increased renal resistive index (RRI) has been recently associated with target organ damage and cardiovascular or renal outcomes in patients with hypertension and diabetes mellitus. However, reference values in the general population and information on familial aggregation are largely lacking. We determined the distribution of RRI, associated factors, and heritability in a population-based study. Families of European ancestry were randomly selected in 3 Swiss cities. Anthropometric parameters and cardiovascular risk factors were assessed. A renal Doppler ultrasound was performed, and RRI was measured in 3 segmental arteries of both kidneys. We used multilevel linear regression analysis to explore the factors associated with RRI, adjusting for center and family relationships. Sex-specific reference values for RRI were generated according to age. Heritability was estimated by variance components using the ASSOC program (SAGE software). Four hundred women (mean age±SD, 44.9±16.7 years) and 326 men (42.1±16.8 years) with normal renal ultrasound had mean RRI of 0.64±0.05 and 0.62±0.05, respectively (P<0.001). In multivariable analyses, RRI was positively associated with female sex, age, systolic blood pressure, and body mass index. We observed an inverse correlation with diastolic blood pressure and heart rate. Age had a nonlinear association with RRI. We found no independent association of RRI with diabetes mellitus, hypertension treatment, smoking, cholesterol levels, or estimated glomerular filtration rate. The adjusted heritability estimate was 42±8% (P<0.001). In a population-based sample with normal renal ultrasound, RRI normal values depend on sex, age, blood pressure, heart rate, and body mass index. The significant heritability of RRI suggests that genes influence this phenotype.

GENCODE: producing a reference annotation for ENCODE.

BACKGROUND: The GENCODE consortium was formed to identify and map all protein-coding genes within the ENCODE regions. This was achieved by a combination of initial manual annotation by the HAVANA team, experimental validation by the GENCODE consortium and a refinement of the annotation based on these experimental results. RESULTS: The GENCODE gene features are divided into eight different categories of which only the first two (known and novel coding sequence) are confidently predicted to be protein-coding genes. 5' rapid amplification of cDNA ends (RACE) and RT-PCR were used to experimentally verify the initial annotation. Of the 420 coding loci tested, 229 RACE products have been sequenced. They supported 5' extensions of 30 loci and new splice variants in 50 loci. In addition, 46 loci without evidence for a coding sequence were validated, consisting of 31 novel and 15 putative transcripts. We assessed the comprehensiveness of the GENCODE annotation by attempting to validate all the predicted exon boundaries outside the GENCODE annotation. Out of 1,215 tested in a subset of the ENCODE regions, 14 novel exon pairs were validated, only two of them in intergenic regions. CONCLUSION: In total, 487 loci, of which 434 are coding, have been annotated as part of the GENCODE reference set available from the UCSC browser. Comparison of GENCODE annotation with RefSeq and ENSEMBL show only 40% of GENCODE exons are contained within the two sets, which is a reflection of the high number of alternative splice forms with unique exons annotated. Over 50% of coding loci have been experimentally verified by 5' RACE for EGASP and the GENCODE collaboration is continuing to refine its annotation of 1% human genome with the aid of experimental validation.

Global Diversity Lines-A Five-Continent Reference Panel of Sequenced Drosophila melanogaster Strains.

Reference collections of multiple Drosophila lines with accumulating collections of "omics" data have proven especially valuable for the study of population genetics and complex trait genetics. Here we present a description of a resource collection of 84 strains of Drosophila melanogaster whose genome sequences were obtained after 12 generations of full-sib inbreeding. The initial rationale for this resource was to foster development of a systems biology platform for modeling metabolic regulation by the use of natural polymorphisms as perturbations. As reference lines, they are amenable to repeated phenotypic measurements, and already a large collection of metabolic traits have been assayed. Another key feature of these strains is their widespread geographic origin, coming from Beijing, Ithaca, Netherlands, Tasmania, and Zimbabwe. After obtaining 12.5× coverage of paired-end Illumina sequence reads, SNP and indel calls were made with the GATK platform. Thorough quality control was enabled by deep sequencing one line to >100×, and single-nucleotide polymorphisms and indels were validated using ddRAD-sequencing as an orthogonal platform. In addition, a series of preliminary population genetic tests were performed with these single-nucleotide polymorphism data for assessment of data quality. We found 83 segregating inversions among the lines, and as expected these were especially abundant in the African sample. We anticipate that this will make a useful addition to the set of reference D. melanogaster strains, thanks to its geographic structuring and unusually high level of genetic diversity.

Fibroblast Growth Factor 8 and its Receptors in The Growth and Angiogenesis of Experimental Breast Cancer . With Special Reference to Thrombospondin-1 as a Novel Target Gene for FGF-8

The growth of breast cancer is regulated by hormones and growth factors. Recently, aberrant fibroblast growth factor (FGF) signalling has been strongly implicated in promoting the progression of breast cancer and is thought to have a role in the development of endocrine resistant disease. FGFs mediate their auto- and paracrine signals through binding to FGF receptors 1-4 (FGFR1-4) and their isoforms. Specific targets of FGFs in breast cancer cells and the differential role of FGFRs, however, are poorly described. FGF-8 is expressed at elevated levels in breast cancer, and it has been shown to act as an angiogenic, growth promoting factor in experimental models of breast cancer. Furthermore, it plays an important role in mediating androgen effects in prostate cancer and in some breast cancer cell lines. We aimed to study testosterone (Te) and FGF-8 regulated genes in Shionogi 115 (S115) breast cancer cells, characterise FGF-8 activated intracellular signalling pathways and clarify the role of FGFR1, -2 and -3 in these cells. Thrombospondin-1 (TSP-1), an endogenous inhibitor of angiogenesis, was recognised as a Te and FGF-8 regulated gene. Te repression of TSP-1 was androgen receptor (AR)-dependent. It required de novo protein synthesis, but it was independent of FGF-8 expression. FGF-8, in turn, downregulated TSP-1 transcription by activating the ERK and PI3K pathways, and the effect could be reversed by specific kinase inhibitors. Differential FGFR1-3 action was studied by silencing each receptor by shRNA expression in S115 cells. FGFR1 expression was a prerequisite for the growth of S115 tumours, whereas FGFR2 expression alone was not able to promote tumour growth. High FGFR1 expression led to a growth advantage that was associated with strong ERK activation, increased angiogenesis and reduced apoptosis, and all of these effects could be reversed by an FGFR inhibitor. Taken together, the results of this thesis show that FGF-8 and FGFRs contribute strongly to the regulation of the growth and angiogenesis of experimental breast cancer and support the evidence for FGF-FGFR signalling as one of the major players in breast cancers.

Promoter region sequence differences in the A and G gamma globin genes of Brazilian sickle cell anemia patients

Fetal hemoglobin (HbF), encoded by the HBG2 and HBG1 genes, is the best-known genetic modulator of sickle cell anemia, varying dramatically in concentration in the blood of these patients. This variation is partially associated with polymorphisms located in the promoter region of the HBG2 and HBG1 genes. In order to explore known and unknown polymorphisms in these genes, the sequences of their promoter regions were screened in sickle cell anemia patients and correlated with both their HbF levels and their βS-globin haplotypes. Additionally, the sequences were compared with genes from 2 healthy groups, a reference one (N = 104) and an Afro-descendant one (N = 98), to identify polymorphisms linked to the ethnic background.The reference group was composed by healthy individuals from the general population. Four polymorphisms were identified in the promoter region of HBG2 and 8 in the promoter region of HBG1 among the studied groups. Four novel single nucleotide polymorphisms (SNP) located at positions -324, -317, -309 and -307 were identified in the reference group. A deletion located between -396 and -391 in the HBG2 promoter region and the SNP -271 C→T in the HBG1 promoter region were associated with the Central African Republic βS-globin haplotype. In contrast, the -369 C→G and 309 A→G SNPs in the HBG2 promoter region were correlated to the Benin haplotype. The polymorphisms -396_-391 del HBG2, -369 SNP HBG2 and -271 SNP HBG1 correlated with HbF levels. Hence, we suggest an important role of HBG2 and HBG1 gene polymorphisms on the HbF synthesis.

Molecular Approaches For Characterization And Determination Of Pathogenicity Of Vibrios With Special Reference To Vibrio Harveyi From Penaeus Monodon Larval Production Systems

The present study focuses on vibrios especially Vibrio harveyi isolated from shrimp (P. monodon) larval production systems from both east and west coasts during times of mortality. A comprehensive approach has been made to work out their systematics through numerical taxonomy and group them based on RAPD profiling and to segregate the virulent from non- virulent isolates based on the presence of virulent genes as well as their phenotypic expression. The information gathered has helped to develop a simple scheme of identification based on phenotypic characters and segregate the virulent from non virulent strains of V. harveyi.

Expression of metallothionein genes I and II in bank vole clethrionomys glareolus populations chronically exposed in situ to heavy metals

The expression of two metallothionein genes (Mt-I and Mt-II) in the liver, kidney, and gonad of bank voles collected at four metal-contaminated sites (Cd, Zn, Pb, and Fe) were measured using the quantitative real-time PCR method (QPCR). Relative Mt gene expression was calculated by applying a normalization factor (NF) using the expression of two housekeeping genes, ribosomal 18S and beta-actin. Relative Mt expression in tissues of animals from contaminated sites was up to 54.8-fold higher than those from the reference site for Mt-I and up to 91.6-fold higher for Mt-II. Mt-II gene expression in the livers of bank voles from contaminated sites was higher than Mt-I gene expression. Inversely, Mt-II expression in the kidneys of voles was lower than Mt-I expression. Positive correlations between cadmium levels in the tissues and Mt-I were obtained in all studied tissues. Zinc, which undergoes homeostatic regulation, correlated positively with both Mt-I and Mt-II gene expression only in the kidney. Results showed that animals living in chronically contaminated environments intensively activate detoxifying mechanisms such as metallothionein expression. This is the first time that QPCR techniques to measure MT gene expression have been applied to assess the impact of environmental metal pollution on field collected bank voles.

Comparative expression analysis of transcription factor genes in the endostyle of invertebrate chordates

The endostyle of invertebrate chordates is a pharyngeal organ that is thought to be homologous with the follicular thyroid of vertebrates. Although thyroid-like features such as iodine-concentrating and peroxidase activities are located in the dorsolateral part of both ascidian and amphioxus endostyles, the structural organization and numbers of functional units are different. To estimate phylogenetic relationships of each functional zone with special reference to the evolution of the thyroid, we have investigated, in ascidian and amphioxus, the expression patterns of thyroid-related transcription factors such as TTF-2/MoxE4 and Pax2/5/8, as well as the forkhead transcription factors FoxQ1 and FoxA. Comparative gene expression analyses depicted an overall similarity between ascidians and amphioxus endostyles, while differences in expression patterns of these genes might be specifically related to the addition or elimination of a pair of glandular zones. Expressions of Ci-FoxE and BbFoxE4 suggest that the ancestral FoxE class might have been recruited for the formation of thyroid-like region in a possible common ancestor of chordates. Furthermore, coexpression of FoxE4, Pax2/5/8, and TPO in the dorsolateral part of both ascidian and amphioxus endostyles suggests that genetic basis of the thyroid function was already in place before the vertebrate lineage. (c) 2005 Wiley-Liss, Inc.

Characterization of the 23S and 5S rRNA genes and 23S-5S intergenic spacer region (ITS-2) of Photobacterium damselae

The 23S ribosomal RNA (rRNA) gene has been sequenced in strains of the fish pathogens Photobacterium damselae subsp. damselae (ATCC 33539) and subsp. piscicida (ATCC 29690), showing that 3 nucleotide positions are clearly different between subspecies. In addition, the 5S rRNA gene plus the intergenic spacer region between the 23S and 5S rRNA genes (ITS-2) were amplified, cloned and sequenced for the 2 reference strains as well as the field isolates RG91 (subsp. damselae) and DI21 (subsp. piscicida). A 100% similarity was found for the consensus 5S rRNA gene sequence in the 2 subspecies, although some microheterogeneity was detected as inter-cistronic variability within the same chromosome. Sequence analysis of the spacer region between the 23S and 5S rRNA genes revealed 2 conserved and 3 variable nucleotide sequence blocks, and 4 different modular organizations were found. The ITS-2 spacer region exhibited both inter-subspecies and inter-cistronic polymorphism, with a mosaic-like structure. The EMBL accession numbers for the 23S, 5S and ITS-2 sequences are: P. damselae subsp. piscicida 5S gene (AJ274379), P. damselae subsp. damselae 23S gene (Y18520), subsp. piscicida 23S gene (Y17901), R damselae subsp. piscicida ITS-2 (AJ250695, AJ250696), P. damselae subsp. damselae ITS-2 (AJ250697, AJ250698).

Systematic review of the frog family Hylidae, with special reference to Hylinae: Phylogenetic analysis and taxonomic revision

Hylidae is a large family of American, Australopapuan, and temperate Eurasian treefrogs of approximately 870 known species, divided among four subfamilies. Although some groups of Hylidae have been addressed phylogenetically, a comprehensive phylogenetic analysis has never been presented. The first goal of this paper is to review the current state of hylid systematics. We focus on the very large subfamily Hylinae (590 species), evaluate the monophyly of named taxa, and examine the evidential basis of the existing taxonomy. The second objective is to perform a phylogenetic analysis using mostly DNA sequence data in order to (1) test the monophyly of the Hylidae; (2) determine its constituent taxa, with special attention to the genera and species groups which form the subfamily Hylinae, and c) propose a new, monophyletic taxonomy consistent with the hypothesized relationships. We present a phylogenetic analysis of hylid frogs based on 276 terminals, including 228 hylids and 48 outgroup taxa. Included are exemplars of all but 1 of the 41 genera of Hylidae (of all four nominal subfamilies) and 39 of the 41 currently recognized species groups of the species-rich genus Hyla. The included taxa allowed us to test the monophyly of 24 of the 35 nonmonotypic genera and 25 species groups of Hyla. The phylogenetic analysis includes approximately 5100 base pairs from four mitochondrial (12S, tRNA valine, 16S, and cytochrome b) and five nuclear genes (rhodopsin, tyrosinase, RAG-1, seventh in absentia, and 28S), and a small data set from foot musculature. Concurring with previous studies, the present analysis indicates that Hemiphractinae are not related to the other three hylid subfamilies. It is therefore removed from the family and tentatively considered a subfamily of the paraphyletic Leptodactylidae. Hylidae is now restricted to Hylinae, Pelodryadinae, and Phyllomedusinae. Our results support a sister-group relationship between Pelodryadinae and Phyllomedusinae, which together form the sister taxon of Hylinae. Agalychnis, Phyllomedusa, Litoria, Hyla, Osteocephalus, Phrynohyas, Ptychohyla, Scinax, Smilisca, and Trachycephalus are not monophyletic. Within Hyla, the H. albomarginata, H. albopunctata, H. arborea, H. boons, H. cinerea, H. eximia, H. geographica, H. granosa, H. microcephala, H. miotympanum, H. tuberculosa, and H. versicolor groups are also demonstrably nonmonophyletic. Hylinae is composed of four major clades. The first of these includes the Andean stream-breeding Hyla, Aplastodiscus, all Gladiator Frogs, and a Tepuian clade. The second clade is composed of the 30-chromosome Hyla, Lysapsus, Pseudis, Scarthyla, Scinax (including the H. uruguaya group), Sphaenorhynchus, and Xenohyla. The third major clade is composed of Nyctimantis, Phrynohyas, Phyllodytes, and all South American/West Indian casque-headed frogs: Aparasphenodon, Argenteohyla, Corythomantis, Osteocephalus, Osteopilus, Tepuihyla, and Trachycephalus. The fourth major clade is composed of most of the Middle American/Holarctic species groups of Hyla and the genera Acris, Anotheca, Duellmanohyla, Plectrohyla, Pseudacris, Ptychohyla, Pternohyla, Smilisca, and Triprion. A new monophyletic taxonomy mirroring these results is presented where Hylinae is divided into four tribes. Of the species currently in Hyla, 297 of the 353 species are placed in 15 genera; of these, 4 are currently recognized, 4 are resurrected names, and 7 are new. Hyla is restricted to H. femoralis and the H. arborea, H. cinerea, H. eximia, and H. versicolor groups, whose contents are redefined. Phrynohyas is placed in the synonymy of Trachycephalus, and Pternohyla is placed in the synonymy of Smilisca. The genus Dendropsophus is resurrected to include all former species of Hyla known or suspected to have 30 chromosomes. Exerodonta is resurrected to include the former Hyla sumichrasti group and some members of the former H. miotympanum group. Hyloscirtus is resurrected for the former Hyla armata, H. bogotensis, and H. larinopygion groups. Hypsiboas is resurrected to include several species groups - many of them redefined here - of Gladiator Frogs. The former Hyla albofrenata and H. albosignata complexes of the H. albomarginata group are included in Aplastodiscus. New generic names are erected for (1) Agalychnis calcarifer and A. craspedopus; (2) Osteocephalus langsdorffii; the (3) Hyla aromatica, (4) H. bromeliacia, (5) H. godmani, (6) H. mixomaculata, (7) H. taeniopus, (8) and H. tuberculosa groups; (9) the clade composed of the H. pictipes and H. pseudopuma groups; and (10) a clade composed of the H. circumdata, H. claresignata, H. martinsi, and H. pseudopseudis groups. Copyright © American Museum of Natural History 2005.

Selection of endogenous genes for gene expression studies in Eucalyptus under biotic (Puccinia psidii) and abiotic (acibenzolar-S-methyl) stresses using RT-qPCR

Background: Rust caused by Puccinia psidii Winter has been limiting for the establishment of new Eucalyptus plantations, as well as for resprouting of susceptible genetic materials. Identifying host genes involved in defense responses is important to elucidate resistance mechanisms. Reverse transcription-quantitative PCR is the most common method of mRNA quantitation for gene expression analysis. This method generally employs a reference gene as an internal control to normalize results. A good endogenous control transcript shows minimal variation due to experimental conditions. Findings. We analyzed the expression of 13 genes to identify transcripts with minimal variation in leaves of 60-day-old clonal seedlings of two Eucalyptus clones (rust-resistant and susceptible) subjected to biotic (P. psidii) and abiotic (acibenzolar-S-methyl, ASM) stresses. Conclusions. For tissue samples of clones that did not receive any stimulus, a combination of the eEF2 and EglDH genes was the best control for normalization. When pathogen-inoculated and uninoculated plant samples were compared, eEF2 and UBQ together were more appropriate as normalizers. In ASM-treated and untreated leaves of both clones, transcripts of the CYP and elF4B genes combined were the ones with minimal variation. Finally, when comparing expression in both clones for ASM-treated leaves, P. psidii-inoculated leaves, ASM-treated plus P. psidii-inoculated leaves, and their respective controls, the genes with the most stable expression were EgIDH and UBQ. The chitinase gene, which is highly expressed in studies on plant resistance to phytopathogens, was used to confirm variation in gene expression due to the treatments. © 2010 Laia et al; licensee BioMed Central Ltd.