Parentage test in broad-snouted caimans (Caiman latirostris, Crocodylidae) using microsatellite DNA

In this study, microsatellite markers, developed for Alligator mississipiensis and Caiman latirostris, were used to assess parentage among individuals from the captive colony of Caiman latirostris at the University of São Paulo, in Piracicaba, São Paulo, Brazil. Many of the females in the colony were full siblings, which made maternal identification difficult due to genotypic similarity. Even so, the most likely mother could be identified unambiguously among offspring in most of the clutches studied. Two non-parental females displayed maternal behavior which would have misled managers in assigning maternity based on behavior alone. This set of variable loci demonstrates the utility of parentage testing in captive propagation programs.

Leptin and hypothalamic gene expression in early- and late-maturing Bos indicus Nellore heifers

We investigated wether early-maturing or late-maturing Bos indicus Nellore heifers produced more leptin mRNA in adipose tissues and altered expression of hypothalamic genes related to leptin signaling. Six prepubertal and six pubertal heifers aged about 34 months and weighing 280 kg to 300 kg each were selected from a population of 100 Nellore heifers. Real-time PCR was used to quantify the expression of the leptin gene (LEP) in adipose tissues and the long isoform of the leptin receptor gene (Ob-Rb), the NK2 homeobox 1 hypothalamic marker gene NKX2-1, the suppressor of cytokine signaling 3 gene (SOCS-3), the neuropeptide Y genes (NPY) and the NPY G-protein coupled receptor genes NPY-Y1 and NPY-Y4 in the hypothalamus. Heifers attaining puberty earlier showed significantly greater LEP expression in adipose tissues (p < 0.05) and there was tissue interaction (p < 0.05). Hypothalamic expression of Ob-Rb, NKX2-1, NPY and SOCS-3 did not differ between groups, but in early-maturing heifers there was a tendency for lower expression of NPY-Y1 (8.3-fold less) and NPY-Y4 (14.3-fold less) compared to late-maturing heifers (p = 0.1). These results suggest that a combination of higher LEP expression, lower NPY-Y1 and NPY-Y4 expression could be a factor in regulating puberty in early-maturing B. indicus heifers.

Effect of growth hormone on fatty acid synthase gene expression in porcine adipose tissue cultures

We describe an efficient in vitro assay to test growth hormone effects on mRNA levels and fatty acid synthase (FAS, EC. 2.3.1.85) activity. Swine adipose tissue explants were long-term cultured in medium containing growth hormone and FAS mRNA levels and enzyme activity were measured. We quantified FAS transcripts by competitive reverse transcriptase PCR (RT-PCR) using total RNA from cultured adipose tissue explants and RT-PCR standard-curves were constructed using a cloned 307 bp segment of native FAS cDNA and a shorter fragment from which a 64 bp (competitor, 243 bp) internal sequence had been deleted. A known amount of competitor was added to each PCR as an internal control and æ-actin transcripts were also measured to correct for differences in total RNA extraction and reverse transcription efficiency. In cultures with added growth hormone FAS mRNA levels decreased 70 percent (p < 0.01) and FAS enzyme activity decreased 22 percent (p < 0.05). These in vitro growth hormone effects were consistent with those observed in vivo, showing that in vitro adipose tissue culture combined with RT-PCR is a useful and accurate tool for studying growth hormone modulation of adipose tissue metabolism. This technique allowed the diagnosis of lower levels of FAS mRNA in the presence of growth hormone and these low levels were associated with decreased FAS activity in the adipose tissue explants.

Caracterização molecular de populações de Euseius citrifolius Denmark & Muma e Euseius concordis (Chant) (Acari: Phytoseiidae) utilizando o seqüenciamento das regiões ITS1 e ITS2 / Molecular characterization of mite populations of Euseius citrifolius Denmark & Muma and Euseius concordis (Chant) (Acari: Phytoseiidae) using sequences of the ITS1 and ITS2 regions

A identificação dos ácaros geralmente é feita com base nas características morfológicas. Entretanto, as evidências morfológicas nem sempre são suficientes para a distinção de espécies muito próximas, levando o taxonomista a considerar aspectos ecológicos, biológicos e, mais recentemente, as características moleculares. A caracterização molecular de populações de Euseius citrifolius Denmark & Muma e E. concordis (Chant) foi realizada com o seqüenciamento da região dos espaçadores internos transcritos ITS1 e ITS2 utilizando-se os primers P1 (5'-AGAGGAAGTAAAAGTCGTAACAAG-3)' e P2 (5'-ATATGCTTAAATTCAGGGGG-3'), localizados nas regiões 18S e 28S do DNA ribossomal, respectivamente. Foram caracterizadas as seguintes populações: E. citrifolius: Arroio do Meio-RS, Campinas-SP e Petrolina-PE; E. concordis: Arroio do Meio, Jaguariúna-SP, Pontes e Lacerda-MT, Petrolina e Viçosa-MG. O seqüenciamento dos ITS1 e ITS2 permitiu separar os grupos de populações de E. citrifolius e de E. concordis. A similaridade entre as seqüências das duas espécies foi superior a 94 por cento. Maior variação entre as populações foi observada no espaçador ITS1 que no espaçador ITS2. O seqüenciamento da região ITS auxilia na identificação de fitoseídeos especialmente em casos de populações que apresentam incompatibilidade citoplasmática e que requeiram informações adicionais.

Mites have usually been identified by their morphological characteristics. However, morphological evidences are not always sufficient to distinguish between closely related species, leading taxonomists to consider additional ecological, biological and, more recently, molecular characteristics in this process. The molecular characterization of populations of Euseius citrifolius Denmark & Muma and Euseius concordis (Chant) was done by sequencing the internal transcribed spacers ITS1 and ITS2 using the P1 (5'-AGAGGAAGTAAAAGTCGTAACAAG-3)' and P2 (5'-ATATGCTTAAATTCAGGGGG-3') primers, located in 18S and 28S regions of the ribosomal DNA. The following populations were characterizate: E. citrifolius: Arroio do Meio-RS, Campinas-SP and Petrolina-PE; E. concordis: Arroio do Meio, Jaguariúna-SP, Pontes e Lacerda-MT, Petrolina and Viçosa-MG. The sequencing of ITS1 and ITS2 allowed the discrimination between the group of populations of E. citrifolius from E. concordis. The similarity between the sequences was more than 94 percent. Most of the variation between populations was observed more in ITS1 than in ITS2. The sequencing of ITS region helps the identification of phytoseiid species when the populations show citoplasmatic incompatibility and need additional information.

Parentage testing and effect of misidentification on the estimation of breeding value in Gir cattle

A correct relationship among sires is essential for an efficient breeding program. Microsatellite markers were used in progeny tests, to assess the paternity of seventy-four probable offspring of nine Gir dairy sires. A 36 percent misidentification rate was observed; however, these errors had minimal effects on the ranking of the nine bulls with regard to their genetic values. The results suggest that paternity tests should be performed in breeding programs, in order to prevent inappropriate paternities from influencing the genetic value of bulls in the future

A new member of the chalcone synthase (CHS) family in sugarcane

Seqüências do banco de dados SUCEST foram analisadas baseado em suas identidades com genes relacionados que codificam enzimas chalcone sintase (CHS). A seqüência da proteína CHS de sorgo (gi-12229613), foi usada para a busca de seqüências no banco de dados do SUCEST, que foram mais similares à CHS. Baseado na similaridade das seqüências de aminoácidos deduzidas, quatorze conjuntos formados por 121 seqüências, foram divididos em dois grupos. O primeiro grupo é mais similar à CHS de sorgo (EC 2.3.1.74) e apresenta a seqüência consenso do sítio ativo de chalcone e stilbene sintase. Análises da expressäo gênica, baseada no número de seqüências de uma dada biblioteca presente em cada grupo, indicaram que neste caso, a maioria das seqüências säo de bibliotecas preparadas de raiz e flores de cana-de-açúcar. O segundo grupo é mais similar à seqüência de aminoácidos deduzida de uma proteína, näo caracterizada, induzida por patógeno (PIl, gi-9855801-) do banco de dados de EST de Sorghum bicolor. Estas duas seqüências de proteínas säo 90 por cento idênticas e tem duas mudanças de aminoácidos no consenso padräo de chalcone e stilbene sintase, e conservam o resíduo de cisteína na posiçäo do sítio ativo. Esta EST näo foi associada a chalcone sintase antes, e apresenta diferenças na seqüência consenso da CHS previamente descrita de sorgo. A maioria das seqüências do segundo grupo säo de bibliotecas de cana-de-açúcar preparadas de raiz e plantas infectadas com Herbaspirillum rubrisubalbicans e Gluconacetobacter diazotroficans. Os resultados indicam que nós identificamos uma chalcone sintase, tal como a encontrada no banco de cDNA de sorgo em uma biblioteca induzida por patógeno, expressa em plantas de cana-de-açúcar, e que ambas as proteínas säo novos membros da super família chalcone e stilbene sintase.

In situ DNA transfer to chicken embryos by biolistics

Ovos fertilizados de galinha foram bombardeados através da técnica de biobalística. A expressäo transiente do gene lacZ, sob o controle do promotor humano citomegalovírus, foi verificada após a transferência in situ. Diferentes níveis de pressäo de gás hélio, vácuo e tipos de partículas foram testados. A taxa de sobrevivência aumentou à medida que a velocidade das partículas diminuíram, entretanto, o nível de expressäo foi menor. Os melhores resultados, combinando taxa de sobrevivência e expressäo, foram obtidos com partículas de ouro, 600 libras por polegada ao quadrado de hélio e 600 mmHg de vácuo. Nestas condiçöes, todos os embriöes bombardeados apresentaram atividade da ß-galactosidase, indicando que esta técnica é eficiente para a transformaçäo de embriöes de galinha.