versión On-line ISSN 1678-4502
Braz. J. Genet. v.20 n.3 Ribeirão Preto sep. 1997
Analysis of polimorphic nucleolus organizer regions in the rainbow
trout (Oncorhynchus mykiss): inheritance mechanism and effects on
(Análise das regiões organizadoras de nucléolo polimórficas em truta
arco-íris (Oncorhynchus mykiss): mecanismo de herança e efeitos no
*1997. Laboratório de Biologia e Genética de Peixes, Departamento de Morfologia, IB,
UNESP, Botucatu, SP, Brasil. Masters thesis. Orienting Professor: Claudio Oliveira.
Cytogenetic analysis performed on the rainbow trout Oncorhynchus mykiss, from the Estação Experimental de Salmonicultura do Instituto de Pesca, Campos do Jordão (SP), showed that the nucleolar organizer regions (NORs) appeared as a single block (N1) or as two blocks (N2) localized in a subterminal position on the long arms of a submetacentric chromosome pair; the N2 form probably may have arisen due to the occurrence of a pericentric inversion involving this chromosomal segment. The cytogenetic analysis allowed the identification of individuals with N1N1 and N1N2 phenotypes in the population while individuals with N2N2 phenotype, supposed to be homozygotes for the inversion, were not encountered. When sex was considered, the frequencies of these phenotypes did not show significant statistical differences ( = 0.06; P < 0.97). The frequency analysis of N1N1, N1N2 and N2N2 phenotypes indicated that the population was not in Hardy-Weinberg equilibrium (= 19.33; P < 0.01). There was a very large number of individuals presenting the phenotype N1N2. Crosses involving four males (two with the phenotype N1N1 and two with the phenotype N1N2) and four females (one with the phenotype N1N1 and three with the phenotype N1N2) were performed, and eight broods were obtained in the F1 generation. No significant differences were found between expected and observed frequencies in the crosses involving N1N1 x N1N2 and N1N2 x N1N1 parental phenotypes. However, significant departures from the expected frequencies were verified in the offspring obtained from crosses involving N1N2 x N1N2 parents. The lack of N2N2 individuals (homozygotes for the inversion) in the population and mainly their absence in the offspring from N1N2 x N1N2 crosses suggest that such condition may be lethal for the rainbow trout. Survivorship rates of embryonic "eyed egg" and fry stage individuals are not different, indicating that a possible lethal effect due to the inversion may act in an earlier phase of the embryonic development.
Publication supported by FAPESP.
Cognition in Turner syndrome (an investigation of 28 subjects through the Bender test and the Piaget scales)
(Cognição na síndrome de Turner (uma investigação de 28 indivíduos através do teste de Bender e da escala de Piaget))
Fátima do Carmo Fonseca Ricardi*
*1996. Instituto de Biociências, USP, São Paulo, SP, Brasil. Masters thesis.
Orienting Professor: P.H. Saldanha.
Piagetian scales (PS) and Bender visual motor gestalt test (BT) were applied to 28 subjects with Turner syndrome (TS) (12 with universal 45,X (TSI) and 16 bearing different types of mosaic or aberrations (TS II), in order to investigate their cognitive performance. Dermatoglyphics were also analyzed to obtain "clues" of embryological changes which possibly come out during the nervous system development to be associated with the cognitive performance of TS subjects.
Statistical analysis of the data was performed through parametric (descriptive statistics, correlation coefficients and stepwise multiple regression analysis) and non-parametric tests (Spearmann and Mann-Whitney/Wilcoxon) by using Statgraphics, Microstat and JMP microcomputer programs.
Dermatoglyphic pattern distribution was not different from those reported in previous studies of TS individuals: ulnar loops (Lu) in the digital patterns for the right hand as well as hypothenar pattern frequencies and finger ridge, a-b, and A-d counts, maximum atd angle, and ulnarity index presented higher values as compared to normal women.
For almost all these dermatoglyphic variables the 45,X subjects presented higher frequencies than the total of TS probands, while the "S" pattern in the hypothenar area and distal loop (Ld) in the I4 interdigital region were the most frequent patterns among mosaic and structural X-chromosome probands.
Their BT levels also confirmed previous researchs revealing a deficit of visual motor perception in TS individuals which could be due to the absence of a cerebral hemispheric lateralization.
On the other hand, cognitive performance of the TS subjects, for the first time investigated through PS, was similar to that of the Brazilian normal population. Nevertheless, parametric and non-parametric statistical analyses showed a significant correlation between the BT scores and the PS values, results suggesting that in some way the development of the visual motor perception and the construction of the elementary logical structures present an incomplete association in TS individuals.
The best performances in the BT and PS were presented by 45,X probands and the worst ones by mosaic and structural TS subjects, so that mosaicism or structural changes of TS karyotypes may have caused a greater disruption in genetic homeostasis than that occurring in the 45,X condition.
The stepwise multiple regression analysis revealed a significant regression of the BT scores and PS levels upon dermatoglyphic parameters. This association could be explained by changes in the common ectodermal origin of the epidermis and the CNS.
TS subjects seem to succeed in compensating their spatial impairments in order to behave in an adaptive way in cognitive and social contacts. Consequently, Genetical Counselling should consider cognitive or psychosocial difficulties presented by TS subjects, by providing an appropriate treatment and orientation to them and to their families.
Publication supported by FAPESP.
Morphological evaluation of the pumpkin (Cucurbita moschata D.)
from Northeast of Brasil
(Avaliação da variabilidade morfoagronômica de abóbora
(Cucurbita moschata D.) do nordeste brasileiro)
*1996. Departamento de Fitotecnia, Universidade Federal de Viçosa, Viçosa, MG, Brasil.
Masters thesis. Orienting Professor: Vicente Wagner Dias Casali.
This work aimed at characterizing morphologically pumpkin (Cucurbita moschata D.) accessions with respect to feasibility and applicability of the descriptor list proposed by Esquinas-Alcazar and Gullick (1983) to discriminate pumpkin accessions from the Germplasm Bank of Cucurbitaceae for the Northeast of Brazil, located at the Agriculture Research Center for the Semi-Arid Tropics (CPATSA-Embrapa) at Petrolina (Pernambuco State). Twenty-two morphological character descriptors were used. Univariate and multivariate analyses such as grouping and canonical analyses were used. Genetic variability was detected among the accessions, and some of them were identified as having specific characteristics to be used in breeding strategies. The grouping analyses showed relatively similar data. The graphical dispersion of the accessions, made up from the scores of the first four canonical variables, indicated accessions B5 and P31 as the most dissimilar. With the canonical analysis, using the method proposed by Singh (1981), the following descriptors were selected: seed and fruit length, biggest fruit diameter, node of the 1st male flower, vine diameter, number of days for flowering 1st female flower, internode, fruit weight, soluble solids, number of days for flowering 1st male flower, and number of seeds/g. However, by means of evaluation of estimation of genotypic correlation coefficient, the following descriptors were discharged: biggest fruit diameter, node of the 1st male flower, internode length, and soluble solids. The descriptor vine diameter is not included in the list of Esquinas-Alcazar and Gullick (1983), being included in this work as proposal of another descriptor, that is sensitive to the detection of pumpkin variability.
Characterization of the larval esterase in the Drosophila mulleri
species complex (Repleta group)
(Caracterização da esterase larval em espécies de Drosophila
do complexo mulleri (grupo Repleta)
Rogério Pincela Mateus*
*1997. Instituto de Biociências, Letras e Ciências Exatas de São José do Rio Preto, SP, Brasil.
Masters thesis. Orienting Professor: Carlos Roberto Ceron.
In the present work we have studied a specific late 3nd-instar larval b-esterase in several Drosophila species of Repleta group. This enzyme, named larval esterase (EST-L), seems to be related to another b-esterase present throughout the life cicle of this insect (EST-8). The purpose of this work was to characterize this specific late 3nd-instar larval esterase in six species of Repleta group, belonging to the mulleri cluster (Drosophila mulleri, D. aldrichi and D. wheeleri) and to the mojavensis cluster (D. mojavenvis, D. arizonae and D. navojoa) of the mulleri complex, as well as in hybrid larvae obtained from mass crosses involving some of these species, and to compare our results with those found in the literature, which indicate that EST-L and EST-8 originated by a tandem genic duplication event with later specialization. We tried to establish biochemical and genetic differences, possible regulatory mechanisms, and physiologic roles of EST-L and EST-8 in this species, that could confirm the above hypothesis. Late third instar larvae were collected directly from the culture medium and were used in electrophoretic analysis on polyacrylamide slab gels. The identification of the esterase bands was achieved using a and b-naphtyl acetates as substrates. Malathion, eserine sulfate, copper sulfate, iodoacetamide, PMSF (a specific serino-protease inhibitor) and E-64 (a diagnostic cysteine-protease inhibitor) were used as specific inhibitors for these enzymes at 1 mM concentration in the presoak and staining procedure, except for E-64 that was used at 5 mM concentration. The isoelectric points of EST-L and EST-8 were determined on 10% polyacrylamide gel containing 5% ampholyte mixture, forming a pH gradient between 6.0 and 8.0. Esterase molecular weights were determined by a non-denaturating method using different gel concentrations. The hybrids were obtained through mass crosses in population boxes, using 200 couples. Five of six species presented homozygous pattern for EST-L, being visualized as one band on the gel. Polymorphism at the EST-L locus was found only in D. navojoa. A slow band pattern (EST-LS) or a fast one (EST-LF) was presented by homozygotes, whereas the heterozygotes showed a three-band pattern. This was also observed for EST-8 locus in this species and in D. mulleri, indicating a dimeric structure for these enzymes. An EST-L/EST-8 interlocus heterodimer was observed in D. mojavensis, D. arizonae and D. navojoa. All species presented the same inhibition behavior for EST-L and EST-8, being the first enzyme inhibited by PMSF, and the second by malathion. Both enzymes showed no sensitivity to the other inhibitors tested. In respect to pI, all species showed values for EST-L and for EST-8 between 6.0 and 7.0. The three mulleri cluster species presented the highest pI values for EST-L (D. mulleri, 6.88; D. wheeleri, 6.59; D. aldrichi, 6.55), while the three mojavensis cluster species presented the lowest ones (D. arizonae, 6.53; D. mojavensis, 6.38; D. navojoa, 6.37). These isoelectric point values differ from those found in literature for the majority of D. melanogaster esterases. Molecular weight estimation based on nondenaturating conditions gave results in the range between 85.000 and 95.000 daltons for both enzymes in all species, with the exception of D. mulleri EST-8 (molecular weight = 99.000 daltons). These values are in agreement with the literature data obtained by molecular filtration for homologous enzymes in D. mojavensis. With regard to the interspecific crosses, asymmetric isolation occurred in the crosses between D. mulleri and D. mojavensis and between D. navojoa and D. mojavensis. In both crosses hybrid larvae were obtained only in the direction involving D. mulleri or D. navojoa females and D. mojavensis males. In the crosses between D. navojoa and D. arizonae, and D. navojoa and D. mulleri, hybrids were produced in both directions. All hybrid larvae presented a three-band pattern for EST-L and for EST-8, with exception of the crosses involving D. navojoa and D. arizonae, in which a broad band was observed, probably a composition of the parent bands and the intermediary hybrid one. It was observed that some hybrid larvae presented no activity for EST-8, a probable consequence of the interspecific condition of these larvae. The esterase pattern found in hybrid larvae has confirmed the quaternary structure of both enzymes as dimers. Our results, as a whole, support the hypothesis that the larval and esterase-8 are really codified by two independent loci. They probably arose by a gene duplication event as old as the Repleta group, as the majority of the species of this group presents both enzymes. EST-L inhibition by PMSF and EST-8 by malathion indicates that an important serine residue is present in the active center of both enzymes.Esterases that have a serine in their active center are classified as serino-esterases. Among the serino-esterases several enzymes with quite different catalytic properties can be found including carboxylesterases, juvenile hormone esterases, lipases, cholinesterases, cholesterol esterases and lisophospholipases. EST-8 inhibition by malathion, an organophosphate, suggests that this enzyme belongs to the carboxylesterase class. EST-L, on the other hand, was not affected by any diagnostic inhibitor tested, and probably belongs to the acetylesterase class. Data in literature show that all D. melanogaster acetylesterases were inhibited by OTFP, a specific inhibitor of lepidopteran juvenile hormone esterase, suggesting the participation of these enzymes in hormone degradation in this species. EST-L could have the juvenile hormone esterase function in species of the Repleta group, since it is an acetylesterase expressed just at the end of the larval phase, period in which the larva-pupa transformations begin. However, we cannot rule out the possibility that EST-L could be a serino-protease displaying some proteolytic activity during the larva-pupa convertion process, as indicated by PMSF inhibition.
Research supported by CNPq.
Publication supported by FAPESP.
Genotoxic evaluation of eugenol and two related phenolic compounds,
isoeugenol and safrole, using somatic mutation and recombination
test in Drosophila melanogaster
(Avaliação da atividade genotóxica do eugenol e de dois compostos
fenólicos correlacionados, isoeugenol e safrol, através do teste para
detecção de mutação e recombinação em células somáticas de
Maria Cristina Munerato*
*1997. Faculdade de Odontologia, Pontifícia Universidade Católica do Rio Grande do Sul,
Porto Alegre, RS, Brasil. Doctoral thesis. Orienting Professor: Heloisa H.R. de Andrade and
In the present study, the potentialities of eugenol (EUG) were investigated using the somatic mutation and recombination test (SMART), in somatic cells of Drosophila melanogaster. In this way, third-stage trans-heterozygous larvae originated from standard crossing (SC) and improved crossing (IC) were subjected to chronic treatment (48 h) with different EUG concentrations. The difference between these two types of crosses lies only on the level of P450 cytochrome-dependent bioactivation, moderate in the SC and high in the IC.
In order to find the possible correlation between chemical structure and genotoxic activity, two other phenolic compounds were analyzed: isoeugenol (ISOE) and safrole (SAF). The former was chosen for being an EUG isomer and metabolite; the latter, for having a well-known carcinogenic activity, was chosen to be the positive control.
The investigation of the EUG genotoxic activity allowed its identification as a pro-genotoxin, once it was ineffective as a promoter of lesions at DNA level in SC, but with positive results in IC, shown by a significant increase in the frequencies of small single spots and twin spots, in two concentrations. Thus, this compound presented a final diagnosis of weak positive genotoxicity, with a predominance of aneugenic and recombinogenic events.
The isomer and metabolic ISOE was diagnosed as a substance devoid of genotoxic action, as it presented negative results in both types of crossing.
The SAF compound, chosen to be the drug-related positive control, was negative in the SC and clearly positive in the IC, with a significant increase only in the frequencies of small single spots - in four out of five doses employed. In this way, the SAF compound was characterized as a pro-genotoxic agent with the predominance of aneugenic activity.
As the EUG and SAF presented positive responses only in the IC, it is possible to conclude that their derivatives produced via P450 were responsible for the outstanding genotoxic activity. Thus, for the EUG there is only one candidate, which is a quinone methide (QM), formed by the oxidation of the allylic chain, named 2-methoxy-4-allylidene-2,5-cyclohexadiene-1-one. As for the SAF, the following metabolites can be listed: QM 2-hydroxy-4-allylidene-2,5-cyclohexadiene-1-one originated from the degradation of 4-allylcatechol, the QM from EUG and the compound 1-hydroxysafrole. The last still needs a second reaction, catalyzed by sulfotransferases to generate the agent supposed to be carcinogenic: 1-sulfooxysafrole.
Regarding the considerations about structure and genotoxic activity, the difference between EUG and ISOE lies in a change of position of an insaturate bond in the side chain in the para position. Thus, the QM resulting from the metabolization of EUG is more stable than that of the ISOE, allowing its permanence inside the cell for long enough to damage the DNA. Concerning the compound SAF, it is the presence of a methylenedioxy group bound to C1 and C2 of the benzene ring that differentiates it from the compound EUG. This favors its oxidation to form a QM.
Finally, the evidence of a weak genotoxic activity, but true for EUG, leads us to suggest its substitution in Dentistry, with the aim of lowering the exposure of humans to the compound, also present in human diet. Apart from this, there are odontologic products more biologically compatible and able to meet satisfactorily the clinical needs in question, with no harm to the treatment carried out.