Thesis Abstracts
Estimates of penetrance using data from monozygotic and dizygotic twin pairs (Estimativas do valor da penetrância utilizando dados de gêmeos monozigóticos e dizigóticos)
Leide de Almeida Praxedes*
*1997. Departamento de Biologia, Instituto de Biociências, Universidade de São Paulo, São Paulo, SP, Brasil. Master's thesis. Orienting Professor: Paulo A. Otto.
Five distinct models were developed to estimate the gene frequency of the dominant allele p and the penetrance value k of polymorphic traits from monozygotic (MZ) and dizygotic (DZ) twin pair data. These models were applied to 23 twin samples in the literature, using the traits `tongue-rolling' ability and `handedness'.
The same parameters were estimated in nine family samples, consisting of couples and their respective offspring, using the models of familial analysis described by Otto et al. (J. Hered. 85: 331-335, 1994).
The results clearly showed that the p and k estimates for the tongue-rolling ability trait were homogeneous for all models and samples; therefore, the distribution of the trait among twin pairs and in family data can be adequately explained by relatively simple monogenic models with incomplete penetrance.
The expected numbers of possible MZ and DZ twin pairs were calculated in pooled samples from the literature using the values of p and k estimated from the family data. Excellent adherence was obtained for all models. However, the methods developed here could not distinguish among the different hypotheses of the five models, a defect also presented by the original method developed by Snyder, which cannot distinguish between simple and multiple recessive traits.
The estimates of p and k for the trait handedness, used to calculate the expected values in the familial method, gave numbers of dominant and recessive individuals that were significantly different from those observed. Also, a generally high degree of heterogeneity for this trait was obtained among the estimates of p and k in the samples with the five models and among the estimates obtained using the three different groups (familial and MZ and DZ twin pairs). Thus, the distribution of the trait handedness cannot be explained satisfactorily by these models.
Research supported by CNPq.
Publication supported by FAPESP.
Cytochemical and morphometric studies of nucleus and stromal fiber distribution in human prostatic lesions (Estudo citoquímico e morfométrico em núcleos e distribuição das fibras do estroma em lesões da próstata humana)
Sebastião Roberto Taboga*
*1997. Departamento de Biologia Celular, Instituto de Biologia, Universidade Estadual de Campinas, UNICAMP, Campinas, SP, Brasil. Doctoral thesis. Orienting Professor: Maria Luiza Silveira Mello and Benedicto de Campos Vidal.
The aim of the present study was to characterize the nuclei and the extracellular matrix in human prostatic lesions, emphasizing the morphology, cytochemistry, karyometry and immunocytochemistry. Transurethral resections and fine needle aspiration of patients presenting prostatic nodular hyperplasia (PNH; 26 cases) or adenocarcinoma (AC; 25 cases) from the northern region of São Paulo State were examined. Twelve of these cases presented no histopathological alterations. These were considered normal and employed as control (Ct). Six- to eight-micrometer thick histological sections were stained by hematoxylin and eosin for analysis of chromatin distribution and karyometry (nuclear area and perimeter and form factor - 200 measurements for each patient), toluidine blue at pH 4.0 for cytochemical study of nucleic acids, Feulgen reaction for DNA, immunocytochemistry for detection of apoptosis (Apoptag kit) and Gömöri's silver impregnation for study of fibrillar collagens.
There were visible differences in the nuclear phenotypes, especially in AC, which showed a marked nuclear pleomorphism. Karyometry showed significant differences in area and perimeter values but not in form factor (Table I), amongst the three classes (Kruskal-Wallis non-parametric statistical test).
The different classifications of the adenocarcinomas, according to the Gleason scale in the combined version, and considering only the first grading, showed no significant differences (P = 0.838 for area, P = 6076 for perimeter and P = 0.9202 for the form factor). There was considerable heterogeneity of the nuclear population. It was concluded that morphometric analysis cannot be used alone to discriminate prostatic lesions, without considering the tumor histoarchitecture.
The use of toluidine blue staining and the Apoptag test demonstrated the absence of apoptotic cells in normal tissue and in adenocarcinomas but they were found in hyperplasias, especially in areas of regression of hyperplastic acini. This suggests that apoptosis represents a factor involved in local recovery of hyperplasia. Its detection could be an additional parameter for follow-up after treatment.
Analysis of silver-impregnated tissue sections showed that collagen fibers of the prostatic stroma respond to external stimuli in their supramolecular dynamics, with modifications in their disposition and fibrillar compactation. There was no modification in collagen molecular integrity in PNH since silver affinity and birefringence were similar to controls. AC had a high degree of non-differentiation. Thin and branched collagen fibers, strongly argyrophilic and birefringent, were detected in regions of cell proliferation. In the adjacent stroma, host of the tumor, hyaline plaques are probable signs of matrix degradation or modification.
Publication supported by FAPESP.
Occupational exposition to chlorinated hydrocarbons in a chemical industry of Cubatão city, São Paulo State: Evolution of clastogenic effects by the micronucleus test (Exposição ocupacional a organoclorados em uma indústria química de Cubatão, São Paulo: Evolução de efeitos clastogênicos pelo teste de micronúcleos)
Lia Giraldo da Silva Augusto*
*1995. UNICAMP-FCM/Hemocentro, Campus da Cidade Universitária "Zeferino Vaz" S/N, Campinas, SP, Brasil. Doctoral thesis. Orienting Professor: Cármino Antônio de Souza.
A study of occupational exposure to organochlorides was initially carried out at a chemical company in Cubatão (São Paulo State). This unit operated from 1967 to 1993, producing carbon tetrachloride as well as perchloroethylene. It is known that the residual waste of these agents is hexachlorobenzene (HCB). Until 1976, this industry also produced pentachlorophenol. In 1992, the occupational public health service examined 21 workers, five of which had enlarged livers. Consequently research involving both workers and ex-workers was made at University of Campinas (UNICAMP). It is known that HCB accumulates in humans and is slowly excreted. The serum blood levels of HCB were examined at the Adolfo Lutz Institute in São Paulo (Residual Pesticides Section). They checked and compared the serum blood levels of 179 workers and ex-workers as well as 10 other workers from a nearby restaurant service and construction company, 18 relatives of employees and another 36 workers from industries in the same area. The employees and ex-employees had higher levels of hexachlorobenzene in their blood than non-employees and relatives.
Eighty-five employees and ex-employees were submitted to a medical and toxicological examination. There seemed to be a very close connection between serum blood levels of HCB and the length of exposure. Those working in the plant were at greatest risk.
The `micronucleus test' was employed in order to evaluate the clastogenical effect of occupational exposure to chlorinated hydrocarbons. Peripheral lymphocytes were stimulated by phytohemagglutinin cytokinesis blocked by cytocalasin B. This process was developed at the HLA histocompatibility lab of UNICAMP's blood center.
From 1993 to 1994, a random sample of 41 individuals, among the 85 employees submitted to medical exams and 28 men from other chemical companies in Cubatão city, who were not exposed to the same substances, was studied and a larger number of micronuclei were observed in the first group. The frequency of micronuclei was not significantly affected by length of exposure or serum blood levels of hexachlorobenzene. Smoking habits also had no influence. This research showed that multiple exposure to carbon tetrachloride, perchloroethylene, hexachlorobenzene and pentachlorophenol had a clastogenic effect in peripheral lymphocytes of workers from the chemical plant. These industrial agents are classified by the IARC (International Agency for Research on Cancer) as being carcinogenic to animals.
Publication supported by FAPESP.
Genetic structure of populations of Cryptocarya moschata Nees & Martius ex Nees (Lauraceae) (Estrutura genética de populações de Cryptocarya moschata Nees & Martius ex Nees (Lauraceae))
Pedro Luís Rodrigues de Moraes*
*1997. Instituto de Biociências, Departamento de Botânica, UNESP, Campus de Rio Claro, Rio Claro, SP, Brasil. Orienting Professor: Dr. Reinaldo Monteiro.
The "canela-nhutinga", Cryptocarya moschata Nees & Martius ex Nees, is an endemic canopy tree species of the Atlantic Rain Forest whose natural populations are evenly distributed throughout its range. However, there is site variation in absolute tree density, which is usually low.
The aim of this work was to obtain information regarding allele distribution and dynamics in natural populations of this species, while characterizing the genetic variability and structure of its populations, its mating system, and its gene flow by means of isozyme markers. Additionally, the spatial genetic structure of one of these populations was analyzed, and the genetic distances of four populations of this species were compared with one population of Cryptocarya aschersoniana Mez, a close taxonomic relative.
Leaf samples of adult individuals from two regions of São Paulo State (Carlos Botelho State Park (N = 141) and Intervales State Park, at Núcleo Saibadela (N = 36), both in the south; and Serra do Mar State Park, at Núcleo de Picinguaba (N = 10), and at Núcleo de Santa Virgínia (N = 27), both in the north) were collected. Seeds were collected from 35 families (N = 692 individuals) and juveniles (N = 60; aged six years) of three populations from diaspores dispersed by muriquis, Brachyteles arachnoides (Primates-Cebidae), at three different locations in the forest at Carlos Botelho State Park. C. aschersoniana individuals (N = 20) were collected at Mata de Santa Genebra, Campinas, SP.
The genetic characterization was performed through six enzymatic systems using starch gel electrophoresis (12% Penetrose 30). Catalase (four loci; eight alleles), acid phosphatase (three loci; six alleles), alkaline phosphatase (four loci; eight alleles), glutamate oxaloacetate transaminase (two loci; six alleles), peroxidase (five loci; 11 alleles), and polyphenoloxidase (two loci; four alleles) enzymatic systems were used. Thus, 20 loci, of which 18 were polymorphic, were analyzed.
The mean numbers of alleles per locus of the progenies, juveniles and adults of C. moschata were 2.0, 1.7 and 2.0, the effective number of alleles were 1.57, 1.43 and 1.66, the percentage of polymorphic loci were 75, 55 and 85%, the mean observed and expected heterozygosities were 0.211/0.313, 0.222/0.227 and 0.323/0.351, and the mean fixation indices were 0.326, 0.022 and 0.079, respectively. These values indicate a high level of gene diversity when compared to other Angiosperm tropical tree species, with the populations of juveniles and adult individuals showing more heterozygotes than the progenies. This indicates that some kind of selection benefiting heterozygotes occurs.
The genetic structure was characterized through Wright's F-statistics estimates, through Nei's subdivided population analysis, and through Cockerham's coancestry coefficients. There was considerable variability within populations for all categories analyzed. The divergences obtained through 
and 
estimates for the adult populations suggest significant genetic drift and/or natural selection effects. However, the lack of divergence between neighboring populations of the northern region, which have no physical isolation, indicates weaker genetic drift and/or natural selection effects when compared to southern populations, which could be physically isolated. Thus, the "stepping-stone" could be a good model for the genetic variability found within this species. The coancestry coefficient for regions did not show any additional allele frequency differences between individuals, suggesting lower levels of gene flow than that found between neighboring populations without isolation.
The estimated gene flow was consistent with the genetic structure found in the adult populations. Although it was relatively low in comparison with those obtained from other Angiosperm tropical tree species, it was sufficient to avoid populational differentiation due to drift. The neighborhood size estimate for the species averaged seven individuals, indicating that the only population with potential for diminished levels of heterozygosity and loss of low frequency alleles is that in the Serra do Mar State Park, Núcleo de Santa Virgínia. Furthermore, preliminary estimates of the neighborhood areas ranged from 4,773 to 36,750 m2 (
14,000 m2), pointing to the need of maintaining at least five reproductive individuals per hectare to sustain the present genetic structure of average populations of C. moschata.
The population of adults from Carlos Botelho State Park showed a nearly random spatial distribution of genotypes, as indicated by the spatial autocorrelation analysis. Seven out of 24 alleles analyzed showed some kind of family structure. Nevertheless, the average for the alleles did not indicate any spatial genetic structure, corroborating the result obtained from the estimated gene flow. This population indicated the need of at least 12 reproductive individuals per hectare to sustain the present genetic structure.
The adult populations of C. moschata showed a pattern characteristic of allogamous species, from the estimates of apparent outcrossing rates 
1.0). The populations of juveniles also appeared to be preferentially allogamous ( 1.0). However, the estimates obtained from progenies revealed discrepancies. From the apparent outcrossing rate ( 0.51), taking all families together, there was an indication that this species has a mixed mating system. By contrast, the multilocus outcrossing rate (
= 0.862 ± 0.028) and the mean unilocus outcrossing rate (
= 0.752 ± 0.053) indicate that the mating system is mainly allogamous. The difference between and was significant, indicating biparental mating. When the multilocus outcrossing rates and the outcrossing-pollen allele frequencies were jointly estimated for each individual tree, the average estimated of the multilocus outcrossing rate was 1.01. This indicates the absence of selfing, as was found for the other categories, and suggests that the difference between the population estimate and the average estimate of each individual tree is caused by matings among relatives. Thus, it is possible to conclude that this species is preferentially allogamous, although there is probably some non-random mating among individuals during each mating cycle, producing progenies without panmixia or inbreeding equilibrium, even when adult individuals are at panmixia equilibrium. The correlation of outcrossed paternal alleles was 37.9%, which could be the results of random outcrossing to a pool of about three near neighbors.
These isozyme data indicate that C. moschata and C. aschersoniana are genetically very similar, in agreement with morphological and anatomical evidence.
Publication supported by FAPESP.
Study of the nucleolus and longitudinal differentiation of Aedes aegypti chromosomes by staining techniques (Estudo do nucléolo e da diferenciação longitudinal dos cromossomos de Aedes aegypti, por técnicas de coloração)
Rita de Cássia de Sousa*
*1998. Departamento de Biologia. IBILCE/UNESP, São José do Rio Preto, SP, Brasil. Master's thesis. Orienting Professor: Hermione E.M. de Campos Bicudo.
We studied the cytogenetics (chromosomal and nucleolar characterization) of brain, testes, ovary and Malpighian tubule tissue of Aedes aegypti. The staining techniques used were lacto-acetic orcein, Feulgen, Ag-staining and C-banding. We analyzed samples of A. aegypti from São José do Rio Preto and Franca, State of São Paulo.
C-banding revealed a single, small heterochromatic body in the interphasic cells of all tissues studied, in both populations. The observations suggested that the heterochromatic regions of all chromosomes are joined to constitute the single C-banded body in interphase cells. The C-banding pattern of the A. aegypti population from São José do Rio Preto differed from that from Franca. Chromosomes 2, 3 and X showed centromeric C-bands. A slightly stained centromeric band in the Y chromosome was observed in some individuals from São José do Rio Preto, but was absent in the Franca population. The X chromosome in both populations and the Y chromosome of all individuals examined from São José do Rio Preto had an intercalary band in one of the arms. This band varied in size, width, and staining intensity. An intercalary band on the secondary constriction of chromosome 3 was also present in some mosquitoes of both populations. This band has not been reported previously. Comparison of our data with data obtained from populations of A. aegypti from Africa, India, Thailand and the USA also showed differences in the presence or absence of centromeric and interstitial bands in sex chromosomes. C-banding analysis of ovary cystocytes showed an association of the nucleolus with the distal region of the intercalary C-band in the X chromosome. This indicates location of the nucleolus organizer region in that part of this band.
Lacto-acetic orcein and Feulgen staining applied to the Malpighian tubules allowed us to observe different levels of condensation and organization of the chromatin and different nuclear sizes, suggesting different variations in cell activity.
Silver staining in interphase cells was restricted to a single nucleolar body in most brain and testis cells and in ovary interstitial cells. Some brain nuclei showed two stained regions. Another pattern of silver staining was detected in brain and testis cells, which exhibited several stained bodies with variations in staining degree and size. The nuclei of the Malpighian tubule cells stained with silver nitrate had a single large nucleolar body, reflecting intense activity in both primary and stellate cells. Silver staining of ovary cystocytes showed two basic patterns: 1) one single compact small body and 2) multiple bodies encompassing large nuclear areas. The percentage staining was as expected for the ratio between the number of oocytes and the number of nurse cells, suggesting the possibility of eventually using such a morphological difference to recognize these cell types in developmental stages. Until now such differentiation is considered impossible. Silver nitrate staining of metaphase chromosomes showed centromeric bands in all of six chromosomes, including X and Y. In some individuals, the X chromosome also showed an intercalary band in the same place where an intercalary C-band was detected, reinforcing the possibility of NOR localization.
Research supported by CNPq.
Publication supported by FAPESP.
Hybrid dysgenesis induced by P and hobo transposable elements in Drosophila melanogaster strains from Brazil (Disgenesia híbrida induzida pelos elementos transponíveis P e hobo em linhagens de Drosophila melanogaster de diferentes regiões brasileiras)
Valéria Cristina Rufo Vetorazzi *
* 1997. Departamento de Biologia, Instituto de Biociências, Letras e Ciências Exatas, IBILCE-UNESP, São José do Rio Preto, SP, Brasil. Master's thesis. Orienting Professor: Cláudia Márcia Aparecida Carareto.
The crossing of different strains of Drosophila melanogaster can result in varying degrees of nonreciprocal hybrid sterility. P-M gonadal dysgenesis occurs when males from a P strain, which have P elements in their genome, are crossed with females from an M strain, without these elements. In the H-E system, gonadal dysgenesis occurs when H males, which have hobo elements, are crossed with E females, without these elements.
Seven natural populations of D. melanogaster, sampled between September 1995 and April 1996 from Porto Alegre (RS), Joinville (SC), Maringá (PR), São José do Rio Preto (SP), Serra do Cipó (MG), Teresina (PI) and São Luís (MA), were studied. Phenotypic tests were made with the purpose of detecting the P and hobo elements in these strains and the degree of hybrid dysgenesis due to mobilization of these elements in the hybrid genome. The occurrence of P and hobo elements was also evaluated by PCR, using the P and hobo element-specific primer sequences. The sizes of the PCR products were as expected in all strains.
Diagnostic crosses, called A, were made by crossing Canton-S (M-E strain) females with males from the experimental strains. These crosses measure the potential to induce dysgenic traits in the germinal tissue of the strains M or E cytotype descendants, when maintained at 29oC (due to P element mobilization) or at 25oC (possibly due to hobo element mobilization). The A* crosses, between females of the experimental strains and males from the strains Harwich (P-E strain) or 23,5/CyL (H-M strain), evaluated the regulatory ability of the strain cytotype to repress the P or hobo elements coming from the father´s genome. Crosses between males and females of the experimental strains, and control crosses for the A and A* diagnostic crosses were also made.
Sixty F1 mated females (that were individually kept in vials for oviposition, at 25oC) and 30 F1 males (mated at both temperatures) were dissected to investigate ovary and testis abnormalities. The F2 progenies from these F1 females were recorded. These data were used to calculate gonadal dysgenesis indexes (GD, Kidwell, 1986) for the F1 hybrids, and fertility and productivity, from four days of oviposition, for the F2 generation of each cross.
For the crosses at 29oC A crosses gave the highest GD values in hybrids with MA (20.0%), followed by SP (19.2%), MG (14.1%) and PI (10.1%) strains, and the smallest values in the PR (9.1%), RS (5.8%) and SC (0.9%) strains. A* crosses gave the highest GD indexes in SC (11.6%) and PI (10.8%) strains, and the smallest in RS (2.5%), MG (1.7%) and SP (0.9%) strains. The control cross (females Canton-S vs. males Harwich) gave 100% gonadal dysgenesis, as expected. Testis dysgenesis percentages were significantly smaller than for ovaries.
Southern strains, characterized by mean annual temperatures varying between 20 and 27oC, and well-defined seasons, presented smaller degrees of gonadal sterility in A crosses than northern strains (MA strain, for example), which come from regions where mean annual temperature is about 36oC. Strains coming from intermediate regions, from Maringá (PR), São José do Rio Preto (SP), Serra do Cipó (MG) and Teresina (PI), with mean annual temperatures varying bewteen 22 and 35oC, had intermediate GDs.
An analysis of similarity was made using the data from the F1 (related to dysgenesis) and F2 (related to productivity) generations. The dendograms showed a strong north-south clinal variation for productivity, and a less clear clinal variation for gonadal dysgenesis, with higher productivity and GD indexes in the northern strains.
The crosses set up at 25oC, with the purpose of mobilizing hobo elements, showed smaller GD values compared to those at 29oC. The A crosses gave highest values for SC (15.8%) and MA (14.28%) strains, followed by MG (9.48%), SP (6.03%), RS (GD = 2.50%) and PR and PI (1.66%). The A* crosses produced smaller GD values, suggesting strong regulatory ability in these strains. The control cross, Canton-S females and 23,5/CyL males, presented a smaller GD than that for P mobilization, with only 25% dysgenic ovaries.
Some generalizations could be made from these data: 1) all seven natural populations sampled from diverse Brazilian regions had P and hobo elements in their genomes, shown by molecular and phenotypic analysis; 2) the seven strains were phenotypically different for both P-M and H-E systems; 3) the strains could be classified as moderate P (MA, MG and SP), Q (PI, PR and RS) and M'(SC) for the P-M system; 4) the P-M system showed a clinal variation in productivity dependent on temperature in the F2 generation, and for gonadal dysgenesis, the latter less well defined than the former; 5) the strains could be classified as H+ (MA and SC) and H0 (PI, MG, SP, PR and RS) for the H-E system; 6) the H-E system did not show the clinal variation observed for the P-M system; 7) the strains were classified as P-H+ (MA), P-Ho (MG and SP), Q-Ho (PI, PR and RS) and M'-H+ (SC) according to the P-M and H-E systems.
Research supported by FUNDUNESP and CNPq.
Publication supported by FAPESP.
Genetic analysis of strains of Providencia alcalifaciens (Enterobacteriaceae) (Análise genética em linhagens de Providencia alcalifaciens (Enterobacteriaceae))
Marise Sobreira Bezerra da Silva*
*1997. Departamento de Microbiologia, Centro de Pesquisas Aggeu Magalhães, FIOCRUZ, UFPE, Recife, PE, Brasil. Master's thesis. Orienting Professor: Alzira Maria Paiva de Almeida.
Providencia alcalifaciens Enterobacteriaceae is a gram-negative bacterium. This organism has been included among the causative agents of diarrhea. However, its occurrence is rather sporadic. Since the mechanism by which P. alcalifaciens causes diarrhea is still not known, we analyzed a collection of 36 P. alcalifaciens strains from the city of Recife, Brazil, for their plasmid content, protein profile and antibiotic susceptibility. Sixteen strains harbored one to five plasmids, from 147 to < 9.5 kb. All the others had no plasmids. Nearly all the strains were susceptible to cephalosporin, chloramphenicol, gentamicin, neomycin and norfloxacin. Four strains were susceptible to all drugs tested. Four strains displayed resistance to tetracycline only. Eleven strains displayed multiple resistance to ampicillin, tetracycline, cotrimoxazol and sulfametoxazol. There was no correlation between plasmid profile and drug resistance. The strains clustered into five groups according to their protein profile. The strains were also screened by PCR for known molecular pathogenicity markers present in other invasive pathogens. However, no amplifications were observed with specific primers directed to psa, ail, invpstb, invent aida senA and irp2 genes. Strains with varied plasmid content, inoculated through the oral route, with or without iron supplementation, proved to be nonpathogenic for mice. None of the tested strains produced ceratoconjunctivitis in rabbits. RAPD analysis allowed us to group the 36 strains into 10 genotypic groups. Taking into account plasmidless strains only, seven genotypic groups still remained. This diversity of genotypic groups is in agreement with the heterogeneity of P. alcalifaciens previously observed. However, other studies are still needed to elucidate the mechanisms of pathogenicity of P. alcalifaciens.
Molecular evolution of gamma 1 and gamma 2 globin genes in atelines (Primates, Platyrrhini) (Evolução molecular dos genes globinas gama 1 e gama 2 em atelíneos (Primates, Platyrrhini))
Carla Maria Marques Meireles*
*1997. Pós-graduação em Genética e Biologia Molecular, Departamento de Genética, Universidade Federal do Pará, Belém, PA, Brasil. Doctoral thesis. Orienting Professor: Maria Paula Cruz Schneider.
Nucleotide sequences corresponding to 9,673 base pairs of aligned positions from the globin genes gamma 1 (
) and gamma (
) were obtained for seven platyrrhine species belonging to all four genera of atelines (Ateles, Lagothrix, Brachyteles and Alouatta). The results showed that is a pseudogene in all genera, due to a 1.8-kb deletion at this locus, confirming the finding by Fitch et al. (1991) in Ateles geoffroyi. Therefore, this deletion is ancient and occurred in the common ancestor of this clade, about 13 million years ago. Furthermore, when the two genes were compared to the single 
gene in Tarsius through patristic distance, was found to be more conserved than in all species. Gene conversion analyses revealed converted stretches in Ateles geoffroyi, Alouatta seniculus and Atelinae, in only one direction, ® . This finding, together with the inactivation, supports the hypothesis that is the preferential and most expressed gene in the majority of platyrrhines. The in Tarsius does not possess the distal CCAAT element, and the proximal element has a point mutation that does not totally inactivate the gene, since the exons presented only synonymous substitutions. Using methods of maximum parsimony, likelihood and distance, the same phylogenetic arrangement was estimated. {Alouatta [Ateles (Lagothrix, Brachyteles)]}, with 100% parsimony bootstrap values for all of these groups. These results are congruent with Harada et al. (1995) and Goodman (1996) for the epsilon and IRBP genes, and thus we agree with the cladistic arrangement: family Atelidae, subfamily Atelinae, tribes Alouattini (Alouatta), and Atelini with the subtribes Atelina (Ateles) and Brachytelina (Lagothrix and Brachyteles).
Research supported by CNPq.
Publication supported by CNPq, NIH and NSF.
Soybean protein quality improvement: Genetic elimination of lipoxygenases and the a' and G4 subunits, use of RAPD-PCR to select genotypes and to identify markers linked to the CGY1 gene (Melhoramento genético da proteína da soja:
Eliminação de lipoxigenases e das subunidades a' e G4, uso de RAPD-PCR na seleção de genótipos e na identificação de marcadores ligados ao gene CGY1)
Marley Marico Utumi*
*1996. Departamento de Fitotecnia, Universidade Federal de Viçosa, Viçosa, MG, Brasil. Doctoral thesis. Orienting Professor: Maurilio Alves Moreira.
This work was carried out on soybean genotypes lacking lipoxygenases and a' and G4 storage protein subunits to determine the effect of this absence on the physical and chemical characteristics of seeds. RAPD-PCR technique was also used to select individuals genetically most similar to the recurrent parent and to identify the RAPD marker linked to the gene which codes for the a' subunit. The genotypes were obtained from crosses among progenitors with large divergences in these characters, followed by nondestructive biochemical analysis. A drastic reduction in the production of hexanal in genotypes without lipoxygenases was observed. This would improve flavor. No decrease in total seed protein content was observed with the elimination of lipoxygenases or a' and G4 subunits. Elimination of the G4 subunit promoted an increase in methionine content, whereas elimination of the a' subunit caused a decrease. Analysis of phenotypic characteristics and RAPD-PCR were used to estimate the genetic distances among individuals of the F2 and F3 populations (selected for the absence of LOX and of the a' and G4 protein subunits) in relation to PB 2,3 (a line without LOX 2 and 3) which was used as recurrent parent. These populations were derived from the following crosses `PBTN' (without LOX 1, 2 and 3) x [`Keburi' (without a') x `UFV 91.751' (without LOX 2 and 3)];. Both the phenotypic characters and the RAPD markers allowed us to discriminate individuals genetically most similar to the PB 2,3 line, with their combined use being more efficient. Use of the bulked segregant analysis technique made it possible to identify an RAPD marker linked in repulsion to the a' gene.
Resistance to anthracnose in the common bean (Phaseolus vulgaris L.): Genetic diversity of Colletotrichum lindemuthianum, inheritance studies and identification of molecular markers (Resistência a antracnose do feijoeiro (Phaseolus vulgaris L.):
Diversidade genética de raças de Colletotrichum lindemuthianum, herança da resistência e identificação de marcadores moleculares)
Ana Lilia Alzate Marin*
*1996. Curso de Genética e Melhoramento, Universidade Federal de Viçosa, Viçosa, MG, Brasil. Doctoral thesis. Orienting Professor: Maurilio Alves Moreira.
Genetic diversity of 22 races of anthracnose fungus (Colletotrichum lindemuthianum) found in Brazil was determined by the RAPD technique. Three groups were defined based on these data. These groups did not correlate with the ones defined by inoculation of the pathogens in differential cultivars. In order to identify molecular markers linked to anthracnose resistant genes, susceptible cultivars Mexico 222, Cornell 49-242, and Michelite were inoculated with three races (64, 73 and 89) which are frequent in central Brazil. Resistant cultivar AB 136 and the populations derived from crosses between this cultivar and the susceptible ones were also inoculated with these fungus races. Ratio was 3:1, in the F2 population from the cross between Michelite and AB 136 (MiA), and Mexico 222 and AB 136 (MeA), the segregation suggesting that a single gene present in cultivar AB 136 determines resistance to races 89 and 64. A segregation ratio of 13:3 was obtained in the F2 from Cornell 49-242 x AB 136 (CoA), indicating that more than one gene determines resistance to race 73. Two DNA bulks from F2 plants contrasting for resistance to each pathogen race were prepared for each population and were amplified with primers which were polymorphic between the respective progenitors. Markers linked to the resistance genes were identified and mapped in the F2 populations. Marker Z04 was mapped at 8.5 cM from the resistance gene (coupling phase) in the MiA cross. Marker Z09 was mapped at 20.4 cM from the resistance gene (repulsion phase) in the same population. Marker Z04 was also linked at 7.5 cM to the gene conferring resistance to race 64 in the MeA cross, suggesting that resistance to races 89 and 64 is determined by a single gene present in AB 136. In cross CoA, markers B20, 06 and N07 were mapped in coupling at 12.8, 43.0 and 79.5 cM, respectively, from the resistance gene, and K07 was linked in repulsion phase, at 28.9 cM.
Genetic studies of common bacterial blight resistance and other agronomic traits in Phaseolus vulgaris L. (Análise genética da resistência ao crestamento bacteriano comum e outras características agronômicas em Phaseolus vulgaris L.)
Rosana Rodrigues*
*1997. Laboratório de Melhoramento Genético Vegetal, Centro de Ciências e Tecnologias Agropecuárias, Universidade Estadual do Norte Fluminense, Campos dos Goytacazes, RJ, Brasil. Doctoral thesis. Orienting Professor: Nilton Rocha Leal.
Many diseases are important limiting factors in dry bean and snap bean yields. Common bacterial blight (CBB), caused by Xanthomonas axonopodis pv. phaseoli, is one of the most serious bacterial diseases for these crops. There is no satisfactory chemical control, therefore resistant cultivars are important. Besides resistance to CBB, other characters, such as pod quality for fresh and processing market, and high yield, must be considered to select parents and segregant populations. Combining abilities of three snap bean genotypes (`Alessa', `Hab 52' and `Hab 198') and two dry bean genotypes (`Bac-6' and `A-794') were determined for disease reaction in leaves and pods, pod number per plant, seed number per plant, pod length, pod diameter, fiber content and plant height. Inoculation with the highly pathogenic isolate CNF 15 was made using a razor blade in leaves and puncture with needles in pods, evaluated seven days after inoculation. Leaves were evaluated by using a 1-5-grade scale and pods by measuring lesion diameter (mm). Diallel analysis was conducted using Griffing's and Gardner and Eberhart's models. General combining ability (GCA) was significant for all characters evaluated. Specific combining ability (SCA) was significant for disease reaction in pods, pod diameter, fiber content and plant height. `Bac-6', `A-794' and `Alessa' were considered superior genotypes for leaf resistance, pod number per plant, seed number per plant, pod length and fiber content. Non-additive effects were predominant in pod reaction and plant height. `Alessa' x `Bac-6', `Alessa' x `A-794' and `Hab 52' x `Bac-6' were the best combinations. Similar results were obtained using the two diallel methodologies. Based on diallel results, a generation mean analysis was made to estimate additive, dominance and epistatic effects for disease reaction using F1, F2 and F3 populations derived from `Alessa' x `A-794'. Additive and dominant gene action were significant for leaf and pod disease reaction. Epistatic effects were only important for leaf resistance.
Study of genetics markers in Yersinia enterocolitica (Enterobacteriaceae) (Estudo de marcadores genéticos em Yersinia enterocolitica (Enterobacteriacea))
Tereza Cristina Arcanjo Leal*
*1996. Departamento de Genética, Universidade Federal de Pernambuco, UFPE, Recife, PE, Brasil. Departamento de Microbiologia, Centro de Pesquisas Aggeu Magalhães, FIOCRUZ, Recife, PE, Brasil. Master's thesis. Orienting Professor: Alzira Ma Paiva de Almeida.
Yersinia enterocolitica, a gram-negative bacterium (Enterobacteriaceae), is responsible for various clinical features in man, which vary from mild diarrhea to mesenteric adenitis or pseudoappendicitis, as well as extraintestinal manifestations. It is widely spread in the environment and in a variety of domestic and wild animal species. Pigs play a prominent role as reservoirs since they harbor the same serotypes found in man. Several isolations of Y. enterocolitica from pigs have been previously made in Brazil. We made a phenotypic and genotypic characterization of pathogenicity factors in 16 Y. enterocolitica serotype O:3 strains, isolated from apparently healthy pigs in Rio de Janeiro, and four strain serotypes O:4, O:5, O:6 and O:13. Among the human strains, six harbored the pYV (± 70 kb) plasmid and accordingly, they were calcium dependent in MOX medium at 37°C. Another pig strain harbored a small cryptic plasmid of about 8.6 kb. Twelve strains showed pesticin sensitivity, and the esculin reaction was negative in 13 strains. PCR analysis of pathogenicity genes using specific primers showed the presence of the ail gene in 14 strains, the irp2 gene in one and the psaA in none. The majority of the pathogenicity markers were absent in the human strains. This was rather surprising, as these strains had been isolated from diarrheic patients.
We did molecular typing of the same Y. enterocolitica strains, which could be grouped into five genotypic profiles, according to the amplification profiles obtained by the RAPD-PCR technique with three random primers. Fifteen pig strains produced identical amplification profiles (genotypic profile 1). Only one produced a different amplification profile (genotypic profile 2). Genotypic profile 1 was also observed in the human O:6 serotype strain. The O:4 and O:13 serotype human strains showed similar amplification profiles with two primers, but with the third primer, each one of them presented a distinct profile. Therefore, these two strains were classified respectively as genotypic profiles 3 and 4. The O:5 serotype human strain produced amplification profile completely different from those observed for other strains (genotypic profile 5). The presence or absence of plasmids did not affect the results of the amplifications. These results show that genetic differences can exist within one serotype and that strains of different serotypes can produce the same profile of amplification with some primers.
Cytogenetic studies of wild rodents for in situ biomonitoring (Utilização de roedores silvestres no biomonitoramento citogenético in situ)
Angela Maria de Souza Bueno*
*1997. Departamento de Biologia e Genética, Instituto de Biociências, USP, São Paulo, SP, Brasil. Master's thesis. Orienting Professor: Maria Nazareth Rabello-Gay.
Two sympatric species of wild rodents (Akodon montensis, 2n = 24 and Oryzomys nigripes, 2n = 62) were evaluated for damage induced by potentially stressing environmental chemicals. The purpose was to evaluate the performance of cytogenetic endpoints as biomarkers. The specimens were collected from three areas, two of which - a rice field and an industrial area - were exposed to environmental impact and a third one was a permanent preservation area. All sites are located at sub-basin IV of the medium valley of the Itajaí River, Timbó, State of Santa Catarina, Brazil. Intraspecific sympatric and allopatric (ISAC) and interespecific (ISC) comparisons were established using as endpoints: 1) frequency of micronucleated erythrocytes in bone marrow and peripheral blood; 2) frequency of cells with chromosomal aberrations; 3) young to mature erythrocyte ratio and mitotic index. The ISAC for A. montensis showed: 1) (a) - in the rice field - possible occurrence of agents inducing genotoxic and cytotoxic effects, both acute and chronic; (b) - in the preservation area - possible occurrence of weak, chronic genotoxic agents; (c) - in the industrial area - possible existence of acute and chronic agents as well as of moderate cytotoxic ones; 2) possible occurrence of spindle poisons in the exposed areas. The ISAC for O. nigripes showed: 1) (a) - in the rice field - possible action of agents causing moderate and chronic genotoxicity and acute and chronic cytotoxicity; (b) - in the preservation area - possible moderate and accidental genotoxic agents; (c) - in the industrial area - possible occurrence of acute and chronic genotoxic agents; 2) (a) - in the rice field - possible existence of clastogens and spindle poisons; (b) - in the preservation area - no evidence for genotoxic agents; (c) - in the industrial area - possible action of spindle poisons. The ISC showed that: 1) the two species differed significantly in the frequencies of micronucleated erytrocytes. As a rule, O. nigripes had the highest values; 2) for clastogenic effects: (a) - in the exposed areas - higher frequencies for O. nigripes; (b) - no significant differences in the preservation area; 3) spindle poison effects: (a) - early chromosomal segregation: higher frequencies for O. nigripes in the exposed areas; (b) - polyploidy: higher frequencies for A. montensis in the rice field and preservation area and for O. nigripes in the industrial area. Results showed that the use of at least two species and the simultaneous analysis of several cytogenetic endpoints can detect and discriminate genotoxic effects of environmental agents. Molecular cytogenetic techniques may improve the method. The extension of the conclusions to human populations is possible.
Research partially supported by FUNCITEC-SC.
Publication Dates
-
Publication in this collection
06 Jan 1999 -
Date of issue
June 1998
