Efficient method for obtaining Lep(ob)/Lep(ob)-derived animal models using adipose tissue transplantations

Background: Leptin-deficient mice (Lep(ob)/Lep(ob), also known as ob/ob) are of great importance for studies of obesity, diabetes and other correlated pathologies. Thus, generation of animals carrying the Lep(ob) gene mutation as well as additional genomic modifications has been used to associate genes with metabolic diseases. However, the infertility of Lep(ob)/Lep(ob) mice impairs this kind of breeding experiment. Objective: To propose a new method for production of Lep(ob)/Lep(ob) animals and Lep(ob)/Lep(ob)-derived animal models by restoring the fertility of Lep(ob)/Lep(ob) mice in a stable way through white adipose tissue transplantations. Methods: For this purpose, 1 g of peri-gonadal adipose tissue from lean donors was used in subcutaneous transplantations of Lep(ob)/Lep(ob) animals and a crossing strategy was established to generate Lep(ob)/Lep(ob)-derived mice. Results: The presented method reduced by four times the number of animals used to generate double transgenic models (from about 20 to 5 animals per double mutant produced) and minimized the number of genotyping steps (from 3 to 1 genotyping step, reducing the number of Lep gene genotyping assays from 83 to 6). Conclusion: The application of the adipose transplantation technique drastically improves both the production of Lep(ob)/Lep(ob) animals and the generation of Lep(ob)/Lep(ob)-derived animal models. International Journal of Obesity (2009) 33, 938-944; doi: 10.1038/ijo.2009.95; published online 16 June 2009

Genome structure of a Saccharomyces cerevisiae strain widely used in bioethanol production

Bioethanol is a biofuel produced mainly from the fermentation of carbohydrates derived from agricultural feedstocks by the yeast Saccharomyces cerevisiae. One of the most widely adopted strains is PE-2, a heterothallic diploid naturally adapted to the sugar cane fermentation process used in Brazil. Here we report the molecular genetic analysis of a PE-2 derived diploid (JAY270), and the complete genome sequence of a haploid derivative (JAY291). The JAY270 genome is highly heterozygous (similar to 2 SNPs/kb) and has several structural polymorphisms between homologous chromosomes. These chromosomal rearrangements are confined to the peripheral regions of the chromosomes, with breakpoints within repetitive DNA sequences. Despite its complex karyotype, this diploid, when sporulated, had a high frequency of viable spores. Hybrid diploids formed by outcrossing with the laboratory strain S288c also displayed good spore viability. Thus, the rearrangements that exist near the ends of chromosomes do not impair meiosis, as they do not span regions that contain essential genes. This observation is consistent with a model in which the peripheral regions of chromosomes represent plastic domains of the genome that are free to recombine ectopically and experiment with alternative structures. We also explored features of the JAY270 and JAY291 genomes that help explain their high adaptation to industrial environments, exhibiting desirable phenotypes such as high ethanol and cell mass production and high temperature and oxidative stress tolerance. The genomic manipulation of such strains could enable the creation of a new generation of industrial organisms, ideally suited for use as delivery vehicles for future bioenergy technologies.

Heat-moisture treatment and enzymatic digestibility of Peruvian carrot, sweet potato and ginger starches

The aim of this work was to study the effects of heat-moisture treatment (27% moisture, 100 degrees C, 16 h) and of enzymatic digestion (alpha-amylase and glucoamylase) on the properties of sweet potato (SP), Peruvian carrot (PC) and ginger (G) starches. The structural modification with heat-moisture treatment (HMT) affected crystallinity, enzyme susceptibility and viscosity profile. The changes in PC starch were the most pronounced, with a strong decrease of relative crystallinity (from 0.31 to 0.21) and a shift of X-ray pattern from B- to A-type. HMTof SP and G starch did not change the Xray pattern (A-type). The relative crystallinity of these starches changed only slightly, from 0.32 to 0.29 (SP) and from 0.33 to 0.32 (G). The extent of these structural changes (PC > SP > G) altered the susceptibility of the starches to enzymatic attack, but not in same order (PC > G > SP). HMT increased the starches digestion, probably due to rearrangement of disrupted crystallites, increasing accessible areas to attack of enzymes. The viscosity profiles and values changed significantly with HMT, resulting in higher pasting temperatures, decrease of viscosity values and no breakdown, i.e., stability at high temperatures and shear rates. Changes in pasting properties appeared to be more significant for PC and SP starch, whereas the changes for G starch were small. Setback was minimized following HMT in SP and G starches.

Identification of protein coding regions using the modified Gabor-wavelet transform

An important topic in genomic sequence analysis is the identification of protein coding regions. In this context, several coding DNA model-independent methods based on the occurrence of specific patterns of nucleotides at coding regions have been proposed. Nonetheless, these methods have not been completely suitable due to their dependence on an empirically predefined window length required for a local analysis of a DNA region. We introduce a method based on a modified Gabor-wavelet transform (MGWT) for the identification of protein coding regions. This novel transform is tuned to analyze periodic signal components and presents the advantage of being independent of the window length. We compared the performance of the MGWT with other methods by using eukaryote data sets. The results show that MGWT outperforms all assessed model-independent methods with respect to identification accuracy. These results indicate that the source of at least part of the identification errors produced by the previous methods is the fixed working scale. The new method not only avoids this source of errors but also makes a tool available for detailed exploration of the nucleotide occurrence.

Isolation and characterization of microsatellite loci in Colletotrichum acutatum, the causal agent of postbloom fruit drop on citrus

From a genomic enriched library, we developed 27 primer pairs from microsatellite flanking sequences for Colletotrichum acutatum, associated to postbloom fruit drop disease on citrus. Loci were characterized using 40 monosporic C. acutatum isolates. Nine primer pairs successfully amplified polymorphic microsatellite regions, with 3-6 alleles per locus, and mean heterozygosities ranging 0.093-0.590 across loci. The suitability of these primers was investigated in four Colletotrichum species as well. These microsatellite markers will be useful for genetic analyses and epidemiological studies of C. acutatum.

Identification of microsatellites markers associated with yield components and quality parameters in sugarcane

Marker assisted selection depends on the identification of tightly linked association between marker and the trait of interest. In the present work, functional (EST-SSRs) and genomic (gSSRs) microsatellite markers were used to detect putative QTLs for sugarcane yield components (stalk number, diameter and height) and as well as for quality parameters (Brix, Pol and fibre) in plant cane. The mapping population (200 individuals) was derived from a bi-parental cross (IACSP95-3018 x IACSP93-3046) from the IAC Sugarcane Breeding Program. As the map is under construction, single marker trait association analysis based on the likelihood ratio test was undertaken to detect the QTLs. Of the 215 single dose markers evaluated (1:1 and 3:1), 90 (42%) were associated with putative QTLs involving 43 microsatellite primers (18 gSSRs and 25 EST-SSRs). For the yield components, 41 marker/trait associations were found: 20 for height, 6 for diameter and 15 for stalk number. An EST-SSRs marker with homology to non-phototropic hypocotyls 4 (NPH4) protein was associated with a putative QTL with positive effect for diameter as also with a negative effect for stalk number. In relation to the quality parameters, 18 marker trait associations were found for Brix, 19 for Pol, and 12 for fibre. For fibre, 58% of the QTLs detected showed a negative effect on this trait. Some makers associated with QTLs with a negative effect for fibre showed a positive effect for Pol, reflecting the negative correlation generally observed between these traits.

Quantitative Trait Loci Mapping and The Genetic Basis of Heterosis in Maize and Rice

Despite its importance to agriculture, the genetic basis of heterosis is still not well understood. The main competing hypotheses include dominance, overdominance, and epistasis. NC design III is an experimental design that. has been used for estimating the average degree of dominance of quantitative trait 106 (QTL) and also for studying heterosis. In this study, we first develop a multiple-interval mapping (MIM) model for design III that provides a platform to estimate the number, genomic positions, augmented additive and dominance effects, and epistatic interactions of QTL. The model can be used for parents with any generation of selling. We apply the method to two data sets, one for maize and one for rice. Our results show that heterosis in maize is mainly due to dominant gene action, although overdominance of individual QTL could not completely be ruled out due to the mapping resolution and limitations of NC design III. For rice, the estimated QTL dominant effects could not explain the observed heterosis. There is evidence that additive X additive epistatic effects of QTL could be the main cause for the heterosis in rice. The difference in the genetic basis of heterosis seems to be related to open or self pollination of the two species. The MIM model for NC design III is implemented in Windows QTL Cartographer, a freely distributed software.

Diversity of endophytic enterobacteria associated with different host plants

Fifty-three endophytic enterobacteria isolates from citrus, cocoa, eucalyptus, soybean, and sugar cane were evaluated for susceptibility to the antibiotics ampicillin and kanamycin, and cellulase production. Susceptibility was found on both tested antibiotics. However, in the case of ampicillin susceptibility changed according to the host plant, while all isolates were susceptible to kanamycin. Cellulase production also changed according to host plants. The diversity of these. isolates was estimated by employing BOX-PCR genomic fingerprints and 16S rDNA sequencing. In total, twenty-three distinct operational taxonomic units (OTUs) were identified by employing a criterion of 60% fingerprint similarity as a surrogate for an OTU. The 23 OTUs belong to the Pantoea and Enterobacter genera, while their high diversity could be an indication of paraphyletic classification. Isolates representing nine different OTUs belong to Pantoea agglomerans, P. ananatis, P, stewartii, Enterobacter sp., and E. homaechei. The results of this study suggest that plant species may select endophytic bacterial genotypes. It has also become apparent that a review of the Pantoea/Enterobacter genera may be necessary.

In silico analysis of candidate genes involved in light sensing and signal transduction pathways in soybean

Several aspects of photoperception and light signal transduction have been elucidated by studies with model plants. However, the information available for economically important crops, such as Fabaceae species, is scarce. In order to incorporate the existing genomic tools into a strategy to advance soybean research, we have investigated publicly available expressed sequence tag ( EST) sequence databases in order to identify Glycine max sequences related to genes involved in light-regulated developmental control in model plants. Approximately 38,000 sequences from open-access databases were investigated, and all bona fide and putative photoreceptor gene families were found in soybean sequence databases. We have identified G. max orthologs for several families of transcriptional regulators and cytoplasmic proteins mediating photoreceptor-induced responses, although some important Arabidopsis phytochrome-signaling components are absent. Moreover, soybean and Arabidopsis gene-family homologs appear to have undergone a distinct expansion process in some cases. We propose a working model of light perception, signal transduction and response-eliciting in G. max, based on the identified key components from Arabidopsis. These results demonstrate the power of comparative genomics between model systems and crop species to elucidate several aspects of plant physiology and metabolism.

Inheritance of growth habit detected by genetic linkage analysis using microsatellites in the common bean (Phaseolus vulgaris L.)

The genetic linkage map for the common bean (Phaseolus vulgaris L.) is a valuable tool for breeding programs. Breeders provide new cultivars that meet the requirements of farmers and consumers, such as seed color, seed size, maturity, and growth habit. A genetic study was conducted to examine the genetics behind certain qualitative traits. Growth habit is usually described as a recessive trait inherited by a single gene, and there is no consensus about the position of the locus. The aim of this study was to develop a new genetic linkage map using genic and genomic microsatellite markers and three morphological traits: growth habit, flower color, and pod tip shape. A mapping population consisting of 380 recombinant F10 lines was generated from IAC-UNA x CAL143. A total of 871 microsatellites were screened for polymorphisms among the parents, and a linkage map was obtained with 198 mapped microsatellites. The total map length was 1865.9 cM, and the average distance between markers was 9.4 cM. Flower color and pod tip shape were mapped and segregated at Mendelian ratios, as expected. The segregation ratio and linkage data analyses indicated that the determinacy growth habit was inherited as two independent and dominant genes, and a genetic model is proposed for this trait.

Real-time Polymerase Chain Reaction Quantification of Porphyromonas gingivalis and Tannerella forsythia in Primary Endodontic Infections

Introduction: Porphyromonas gingivalis and Tannerella forsythia are anaerobic bacteria commonly involved in root canal infections. Although previous investigations have assessed these species by strictly qualitative approaches, accurate determination of their cell levels by a sensitive quantitative technique may contribute with additional information regarding relevance in pain of endodontic origin. Method: The root canal levels of P gingivalis, T forsythia, and total bacteria were investigated by a quantitative polymerase chain reaction (PCR) assay based on unique copy molecular markers. A total of 32 symptomatic (n = 14) and asymptomatic (n = 18) cases of endodontic infections were analyzed. Root canal samples were collected; genomic DNA was extracted and submitted to SYBR Green I real-time PCR targeting the rgpB (P gingivalis), bspA (T forsythia), and rpoB (total bacteria) single copy genes. Results: Overall, R gingivalis, T forsythia, and the coexistence of both species were encountered in 28%, 66%, and 22% of the subjects, respectively. P gingivalis and T forsythia levels ranged from 5.65 x 10(-6) to 1.20 x 10(-2) and from 5.76 x 10(-6) to 1.35 x 10(-1). T forsythia was highly prevalent and numerous in the study groups, whereas P gingivalis was moderately frequent and less abundant, displaying 19-fold lower average levels than the former. Conclusions: The endodontic levels of P gingivalis and T forsythia, individually or in conjunction, did not display significant associations with the manifestation of pain of endodontic origin. (J Endod 2009,35:1518-1524)

Development and characterization of microsatellite markers for turmeric (Curcuma longa)

P>Curcuma longa L. is a sterile, triploid, vegetatively-propagated crop cultivated mainly in Southeast Asia. When dried rhizomes are ground, the resulting yellow powder is used by the food industry as a natural food dye. Moreover, many pharmacological compounds have broadened the commercial application of the crop. However, conventional breeding is difficult and hence, improvement has been limited to germplasm selection. To better utilize the germplasm collections and facilitate genotype selection, a total of 17 polymorphic microsatellite loci were developed using a CT/GT/CTT enriched genomic library. All microsatellites resulted in amplified PCR products, showing a banding pattern of 2-11 polymorphic bands per locus, enabling genotype discrimination. These results can be used in further studies aimed at characterizing C. longa genetic resource collections and also to improve breeding strategies.

Genomics of pyrrolnitrin biosynthetic loci: evidence for conservation and whole-operon mobility within Gram-negative bacteria

Pyrrolnitrin (PRN) is a tryptophan-derived secondary metabolite produced by a narrow range of Gram-negative bacteria. The PRN biosynthesis by rhizobacteria presumably has a key role in their life strategies and in the biocontrol of plant diseases. The biosynthetic operon that encodes the pathway that converts tryptophan to PRN is composed of four genes, prnA through D, whose diversity, genomic context and spread over bacterial genomes are poorly understood. Therefore, we launched an endeavour aimed at retrieving, by in vitro and in silico means, diverse bacteria carrying the prnABCD biosynthetic loci in their genomes. Analysis of polymorphisms of the prnD gene sequences revealed a high level of conservation between Burkholderia, Pseudomonas and Serratia spp. derived sequences. Whole-operon- and prnD-based phylogeny resulted in tree topologies that are incongruent with the taxonomic status of the evaluated strains as predicted by 16S rRNA gene phylogeny. The genomic composition of c. 20 kb DNA fragments containg the PRN operon varied in different strains. Highly conserved and distinct transposase-encoding genes surrounding the PRN biosynthetic operons of Burkholderia pseudomallei strains were found. A prnABCD-deprived genomic region in B. pseudomallei strain K96243 contained the same gene composition as, and shared high homology with, the flanking regions of the PRN operon in B. pseudomallei strains 668, 1106a and 1710b. Our results strongly suggest that the PRN biosynthetic operon is mobile. The extent, frequency and promiscuity of this mobility remain to be understood.

SAGE transcript profiling of the juvenile cambial region of Eucalyptus grandis

Despite the importance of Eucalyptus spp. in the pulp and paper industry, functional genomic approaches have only recently been applied to understand wood formation in this genus. We attempted to establish a global view of gene expression in the juvenile cambial region of Eucalyptus grandis Hill ex Maiden. The expression profile was obtained from serial analysis of gene expression (SAGE) library data produced from 3- and 6-year-old trees. Fourteen-base expressed sequence tags (ESTs) were searched against public Eucalyptus ESTs and annotated with GenBank. Altogether 43,304 tags were generated producing 3066 unigenes with three or more copies each, 445 with a putative identity, 215 with unknown function and 2406 without an EST match. The expression profile of the juvenile cambial region revealed the presence of highly frequent transcripts related to general metabolism and energy metabolism, cellular processes, transport, structural components and information pathways. We made a quantitative analysis of a large number of genes involved in the biosynthesis of cellulose, pectin, hemicellulose and lignin. Our findings provide insight into the expression of functionally related genes involved in juvenile wood formation in young fast-growing E. grandis trees.

Orchid fleck symptoms may be caused naturally by two different viruses transmitted by Brevipalpus

Brevipalpus-transmitted viruses (BTV) cause chlorotic, necrotic and/or ringspot lesions in leaves and stems of orchids, citrus, coffee and several other plant species. There are two different types of BTVs, the nuclear and the cytoplasmic, based on maturation locale in the cell and particle morphology. The orchid fleck virus (OFV) is a BTV that infects orchids. Its short rodlike particles are 32-40 nm in diameter, 100-150 nm in length. OFV is found in the nucleus and is associated with intranuclear electronlucent viroplasms. In 1999, transmission electron microscopy analysis revealed a distinct type of virus causing orchid fleck symptoms. The bacilliform particles, 70-80 nm in diameter and 110-120 nm in length, induced electron-dense viroplasm inclusions in infected cells and resembled the cytoplasmic type associated with BTV, such as the citrus leprosis virus C. Our objective in the present study was to verify whether the cytoplasmic type virus found in orchids could be amplified using primers for other cytoplasmic BTVs, such as CiLV-C and Solanum violaefolium ringspot virus (SvRSV). Additionally, we aimed to differentiate the two BTVs found in orchids: the nuclear and the cytoplasmic types of OFV using microscopy and molecular and serological tools. This virus was not amplified by the CiLV-C and SvRSV primers, and neither the molecular nor the serological tools available to the OFV diagnosis reacted with it, demonstrating that they are definitely different viruses.