Print version ISSN 1415-4757
Genet. Mol. Biol. vol. 21 n. 4 São Paulo Dec. 1998
Genetic analysis of hybrid sweet pepper fruit traits
(Análise genética de caracteres de fruto em híbridos de pimentão)
Arlete Marchi Tavares de Melo*
*1997. Instituto de Genética. Escola Superior de Agricultura "Luiz de Queiroz", USP, Piracicaba, SP, Brasil. Doctoral thesis. Orienting Professor: Cyro Paulino da Costa.
Three-way hybrid sweet pepper (Capsicum annuum L.) performance was studied with evaluation of heterosis. Yield and fruit trait correlations were determined. The hybrids were obtained from two semiconical cultivars, Myr-29 (group 1) and Magda (group 2), crossed with 17 single hybrids, 15 "Lamuyo type" and two semiconical type. All 30 three-way hybrids were evaluated comparing cv. Myr-29, cv. Magda and the standard Magali hybrid. They were grown in a green polyethylene house at the SVS of Brazil Experimental Station, located in Paulínia, SP in 1995. The experimental design used was a completely randomized design with five replications. The following traits were evaluated: total yield per plant, total number of fruit per plant, average fruit weight, average weight until tenth fruit, early yield weight, early yield fruit number, commercial fruit classification, fruit quality index, fruit wall thickness, fruit length, fruit width, length/width ratio, fruit shape, number of locules per fruit, and number of days to flowering.
The main conclusions were: 1) the best three-way hybrids were: 24 (Reinger x Myr-29), 21 (Pacific x Myr-29), 6 (Mikalor x Myr-29), 11 (Vidi x Myr-29), 17 (Sidor x Myr-29) and 15 (Lamuyo x Myr-29). They were equivalent or superior to the Magali hybrid; 2) expressive heterosis was detected in relation to the best local cultivar Myr-29 for the following traits: total fruit yield up to 46.07% improvement, early yield weight up to 154.74%, and early yield fruit number up to 192.31%; 3) three-way hybrids outperformed the standard Magali for wall thickness; 4) four three-way hybrids had increased premium fruit quality; 5) positive genotypical correlations were found between total yield and total fruit number, fruit weight, weight until tenth fruit, and wall thickness; 6) the three-way hybrids made from cv. Myr-29 crossing had better combining ability than those made with cv. Magda, and 7) differences among three-way hybrids were noticeable for fruit traits but not for yield components.
Research supported by CNPq and CAPES.
Publication supported by FAPESP.
Esterase patterns of Aedes aegypti and Aedes albopictus
(Padrão de esterases em Aedes aegypti e Aedes albopictus)
Alba Regina de Abreu Lima-Catelani*
* 1996. Departamento de Biologia, Instituto de Biociências, Letras e Ciências Exatas, IBILCE-UNESP, São José do Rio Preto, SP, Brasil. Master's thesis. Orienting Professor: Hermione Elly Melara de Campos Bicudo.
Aedes aegypti from Marília and São José do Rio Preto, SP, and A. albopictus samples from Taubaté, SP, were collected. Polyacrylamide gel electrophoresis was used to study esterase patterns of developmental stages L2, L3 and L4, pupae and adult males and females of both species.
The zymograms obtained showed 22 esterase bands in A. aegypti samples from Marília, 19 in those from São José do Rio Preto and 20 in A. albopictus. These numbers are greater than those previously found in these species by other authors. Although A. aegypti and A. albopictus are considered to be close species, their esterase bands did not correspond.
The number of esterase bands was greater in the larval stages of both species. A decrease of 50% in the number of bands from larval to pupal stage and of 30% from pupae to adults was observed in A. aegypti from Marília. In the São José do Rio Preto populations this decrease was 40% and 20%, and 26% and 14%, respectively, in A. aegypti and A. albopictus.
Among the 23 bands of A. aegypti, five were b-esterases and 18 were a-esterases. In A. albopictus, 17 were a-esterases and 3 were b-esterases. The b-esterases were found exclusively in the anodic region of the gel and were more frequent in larvae than in pupae. The a-esterases were found all in gel regions, in every developmental stage.
Highly frequent bands that occurred during all developmental stages are probably related to basic biological functions. Band 1 from A. aegypti appeared in 100% of individuals in every stage analyzed in both populations. A parallelism seems to exist between band 1 and band EST-6 described in the literature.
Stage-specific esterases were detected, including a bands 3 to 5 and 6 and 9 from A. aegypti, present in the Marília and São José do Rio Preto populations, and bands 14, 18 and 20, of A. albopictus. All of these bands are characteristic of larval stages. Bands 15 and 17 were exclusively found in A. aegypti adults from Marília and occurred in both sexes. These differences show that there is variation in esterase synthesis during development in both species. In the pupal stage, exclusive bands were also found, but their frequencies were low, making their specificity uncertain.
Only one band was found exclusively in the adult stage, restricted to A. aegypti males from Marília, it had a high frequency and is assumed to be a male-specific band.
There were individual patterns of band combinations. The 4th instar larvae had the highest number in both species, but at similar low frequencies, suggesting random combinations.
Pupal stages of A. aegypti showed 38 patterns in Marília and 34 in São José do Rio Preto populations. Twelve patterns were shared by both populations; the pattern constituted by bands 1, 12, and 16 was the most frequent in both populations, which suggests that this is an important combination of esterases in this developmental stage. Most of the combinations, shared or not, were found at low frequencies in A. aegypti as well as in A. albopictus. The latter species had a higher number of patterns (119), but the most frequent was found in only four individuals.
In A. aegypti adults from Marília, the number of patterns was also higher than in those from São José do Rio Preto (28 patterns in females and 29 in males from the first locality and 16 in females and 14 in males from the second). In A. albopictus adults, 27 patterns in females and 38 in males were observed. This difference in number of patterns observed among populations might be due to selection acting more strongly in external environments than in the laboratory.
Inhibitors used in an attempt to classify the biochemical nature of the esterases detected in A. aegypti and A. albopictus, showed that none of them can be classified as acetylesterase or arylesterase.
The inhibition pattern showed by bands 1 to 7, 18 and 20 of A. aegypti indicated that they are carboxylesterases. Several studies in literature involving a variety of organisms have shown that carboxylesterase activity is greater in strains resistant to organophosphates and carbamates than in sensitive strains. The results of the inhibition tests reinforce the idea that band 1 may be related to EST-6.
Bands 12 and 14 of A. aegypti and bands 1 to 6 and 17 of A. albopictus showed inhibition patterns characteristic of cholinesterase. They were classified as cholinesterase only in the latter species. Band 13 of A. aegypti and bands 8 to 13 of A. albopictus had cholinesterase characteristics, although they were only slightly inhibited by malathion. They may be malathion-resistant cholinesterase. Sodium fluoride was more specific for inhibition of the larval bands, which suggests that this substance could be used in the control of A. aegypti and A. albopictus.
In conclusion A. aegypti and A. albopictus had esterase band variation during development. There were also differences between A. aegypti samples maintained in the laboratory and those collected in nature.
Publication supported by FAPESP.
Cytogenetic characterization of some species and interspecific hybrids in Passiflora
(Caracterização citogenética de algumas espécies e híbridos interespecíficos de Passiflora)
Marta Dias Soares-Scott*
*1998. Departamento de Biologia Celular, Instituto de Biologia, Universidade Estadual de Campinas, UNICAMP, Campinas, SP, Brasil. Master's thesis. Orienting Professor: Dr. Shirlei Maria Recco-Pimentel.
The most widely cultivated commercial species of passion fruit Passiflora edulis f. flavicarpa is susceptible to diseases. Interspecific hybridization has been the best alternative in the search for disease resistance in genetic breeding programs.
Three species (P. edulis f. flavicarpa, P. setacea and P. incarnata) were used as parents and two interspecific hybrids were produced: P. edulis f. flavicarpa x P. setacea (sexual hybrid) and P. edulis f. flavicarpa + P. incarnata (somatic hybrid). The parental species and sexual hybrid karyotypes had 2n = 18 chromosomes and the somatic hybrid 2n = 36, proving the origin by protoplast fusion. The species karyotypes had similar morphology. Metacentric and submetacentric chromosomes always had a secondary constriction. All the species and hybrids studied had a large number 4 chromosome pair, with a secondary constriction in the apical position of the long arm. P. setacea and its hybrid P. edulis f. flavicarpa x P. setacea had secondary constrictions in three pairs of chromosomes. The somatic hybrid had secondary constrictions in four pairs of chromosomes.
P. incarnata had the greatest absolute mean length and the sexual hybrid had the greatest total karyotype length. The somatic hybrid had a two times greater total karyotype length.
Meiotic analysis of the hybrids showed irregularities during microsporogenesis. Homology was greatest between P. edulis f. flavicarpa and P. setacea as demonstrated by the number of interspecific hybrid bivalents. Both hybrids gave evidence of genetic recombination by multivalent formation. Micronuclei were observed in meiosis II in the somatic hybrid. As a consequence there were more than four microspores in the tetrads phase and pollen grains varied in size.
Pollen viability was low in both hybrids. The meiotic irregularities observed may be responsible for the low level of pollen viability.
Publication supported by FAPESP.
Karyotypical evolution of marsupials from the family Didelphidae
(Evolução cariotípica de marsupiais da família Didelphidae)
* 1998. Departamento de Biologia, Instituto de Biociências, Universidade de São Paulo, São Paulo, SP, Brasil. Ph.D. Thesis. Orienting Professor: Dr. Angela M. Vianna-Morgante.
We made chromosomal analyses of eight species of American marsupials from the Didelphidae, encompassing the three diploid numbers known for this family (2n = 14, 18 and 22). Comparative analyses of the G- and C-banding patterns of Philander opossum, Didelphis marsupialis (2n = 22), Monodelphis domestica (2n = 18), Micoureus cinereus, Marmosops incanus, Caluromys philander and Metachirus nudicaudatus (2n = 14) allowed us to establish homologies between all autosomal arms in the karyotypes with the three different diploid numbers. Robertsonian rearrangements, pericentric inversions and a variable amount of pericentromeric constitutive heterochromatin explain the differences between karyotypes. Interspecific variation in the size of the sexual chromosomes was due to differences in heterochromatic content. G- and C-banding patterns of M. incanus and M. nudicaudatus were described for the first time.
Analyses of nucleolus organizer regions (NORs) using fluorescent in situ hybridization (FISH) with an rDNA probe and silver staining allowed us to confirm the variability in the number and location of NORs, already described in the literature. Furthermore, our data showed conservation of NORs in one autosomal pair in the three karyotypes (2n = 14, 18 and 22). In Monodelphis (2n = 18), the NOR in the X chromosome was not inactivated in females and a regulation mechanism based on overexpression of the NOR in the male X chromosome was found.
Genomic comparisons through FISH with total genomic DNA showed extensive homologies of euchromatic segments between species with 2n = 14 (M. incanus and M. cinereus) and between those and one species with 2n = 22 (P. opossum). The basic difference between these genomes was the repetitive DNA sequences of the autosomal constitutive heterochromatin, that seemed to be species specific.
In situ methylation patterns were analyzed with a monoclonal antibody against anti-5-methylcitosine. Hypomethylation of the constitutive heterochromatin was evidenced in four species (M. incanus, M. cinereus and M. nudicaudatus, 2n = 14, and P. opossum, 2n = 22), a pattern strikingly different from the hypermethylation of heterochromatic regions of other vertebrates. Hypermethylation of heterochromatin was found in C. philander (2n = 14), M. domestica and Monodelphis sp. (2n = 18), and correlated with the GC richness of these segments. In C. philander and M. domestica, the active X chromosome was hypermethylated, compared to the inactive X.
FISH with a telomeric sequence in species with the three diploid numbers revealed interstitial telomeres in five autosomal pairs of Marmosops incanus (2n = 14) and in one pair of Monodelphis domestica (2n = 18). This finding led us to propose a new hypothesis for karyotypical evolution in Didelphidae, based on reduction of diploid numbers through chromosome fusions in a hypothetical ancestral karyotype with at least 24 chromosomes (22 autosomes and the sexual pair). This idea challenges the long accepted notion that chromosomal evolution in marsupials was mainly based on centric fissions that occurred in an ancestral basic karyotype with 2n = 14.
Publication supported by FAPESP.
Cytogenetics studies of anurans of the family Leptodactylidae (Amphibia), with differential staining techniques
(Estudos citogenéticos de anuros da família Leptodactylidae (Amphibia), com técnicas de coloração diferencial)
Ana Paula Zampieri Silva*
*1998. Departamento de Biologia, Instituto de Biociências, Universidade Estadual Paulista, UNESP, Rio Claro, SP, Brasil. Master's thesis. Orienting Professor: Dr. Sanae Kasaharas.
Nine species of anurans belonging to the family Leptodactylidae from various localities were analyzed cytogenetically. The chromosome preparations were made with Giemsa staining, NOR banding, C banding, and replication banding by incorporation of 5-bromodeoxyuridine (BrdU).
Leptodactylus fuscus, L. notoaktites, L. ocellatus, and L. labyrinthicus were studied. The first two are included in the fuscus morphological group and the others in the ocellatus and pentadactylus groups, respectively. All the species had a diploid number of 2n = 22 chromosomes, and a similar karyotype made up of metacentric and submetacentric chromosomes. The eighth pair was the marker in all species, with a secondary constriction in the short arm of the chromosomes. The NOR stained in the region of the secondary constriction. Despite the karyotypic similarity among the four species of Leptodactylus, variations were observed in the C banding pattern, between the different species and within several populations of the species L. fuscus and L. ocellatus. C banding confirmed that the heteromorphism present in the karyotype of specimens of L. ocellatus from Guaratuba, PR, was in fact caused by a pericentric inversion. The pattern of replication bands obtained by incorporation of BrdU was similar in the species L. notoaktites, L. ocellatus, and L. labyrinthicus.
Physalaemus biligonigerus, P. cuvieri, P. olfersii, and P. crombiei were also studied. The first two belong to the biligonigerus and cuvieri morphological groups, respectively, and the other two to the signifer group. All the species had a diploid number of 2n = 22 chromosomes and a similar karyotype made up of metacentric and submetacentric chromosomes, with the exception of P. crombiei which had a pair of acrocentric chromosomes. The eighth pair was the marker in P. biligonigerus, P. cuvieri, and P. crombiei, showing the secondary constriction, whereas P. olfersii had a secondary constriction in pairs 3 and 4. A heteromorphism was observed on pair 11 in P. cuvieri from Rio Claro, SP, with one of the homologues presenting an atypical morphology, with various secondary constrictions. All the chromosomes with secondary constriction were stained by NOR technique, including one of the chromosomes of pair 11 of P. cuvieri, with three NORs along its arms. C banding technique showed slightly different patterns of distribution of the constitutive heterochromatin in the karyotypes of P. biligonigerus and P. cuvieri. In P. cuvieri C banding results corroborated the difference between the karyotypes of individuals from distinct populations, first seen with Giemsa and NOR staining.
Cycloramphus boraceiensis was the only species with a karyotype of 2n = 26 chromosomes. It is constituted mainly by metacentric and submetacentric chromosomes and only one acrocentric pair. This marker pair has a secondary constriction which is stained by the NOR technique. The C banding technique showed heterochromatic blocks only in the large and intermediate-sized chromosomes, being absent in the small ones.
Heteromorphic sex chromosomes were not observed. Absence of a morphologically differentiated sex chromosome pair was confirmed by the meiosis data.
The lymphocyte culture technique gave satisfactory results in terms of quantity and quality of the metaphases. This procedure provided superior NORs and C banding, and also enabled the use of in vitro BrdU treatment, giving good quality replication bands.
Research supported by CNPq and FAPESP.
Publication supported by FAPESP.
Kinetics of cell proliferation and chromosomal instability in different cell lines from individuals with chromosomal mosaicism
(Cinética da proliferação celular e instabilidade cromossômica nas diferentes linhagens celulares de indivíduos com mosaicismo cromossômico)
*1998. Departamento de Morfologia, Escola Paulista de Medicina, UNIFESP, Botucatu, SP. Master Thesis, Orienting Professor: Maria Isabel Melaragno.
Patients with chromosomal mosaicism present two or more distinct cell lines. The relative proportions of these cell lines may change with age of the patient. The few known cases concerning mosaic patients at different ages predominantly show a selective advantage for the normal cell line. Mosaic patients, in whom normal and aneuploid cells grow side by side, provide useful material for the cytogenetic comparison of the different cell populations because they are under the influence of the same endogenous and exogenous factors.
We studied eight mosaic patients that had been karyotyped before: one had Turner syndrome (45,X/46,XX), six had Down syndrome (two 47,XX,+21/46,XX and four 47,XY,+21/46,XY) and one had Klinefelter syndrome (47,XXY/46,XY). Lymphocytes were cultured for 48, 72 and 96 h. 5-Bromodeoxyuridine (BrdU) was used for sister chromatid differentiation. We prepared exposed cultures or not to mitomycin C (MMC), a clastogenic agent.
We evaluated the karyotype and cell proliferation kinetics through analysis of the frequency of first- , second- and third- or fourth-division metaphases in the cultures. Spontaneous and induced sister chromatid exchange (SCE) frequencies and acquired structural chromosome aberrations were also compared between cell lines.
Although all patients showed a decrease in the aneuploid cell line from the previous karyotype analyses, the decrease was significant only in the six patients with trisomy 21, indicating that selection had predominantly favored the normal cell line.
None of the patients showed a statistically significant difference between the normal and aneuploid cell lines in terms of cell kinetics, SCE or acquired structural chromosome aberration frequencies.
Therefore, our data do not allow us to attribute preferential cell line selection with age, nor were there significant differences in cell cycle duration or chromosomal stability between normal and aneuploid cell lines.
Publication supported by FAPESP.
The effect of stress on triatomine Malpighian tubule cell nuclei
(Efeito do estresse nos núcleos das células dos túbulos de Malpighi de triatomínios)
Patrícia Martins Casseb Hassan*
*1997. Departamento de Biologia, Instituto de Biociências, Letras e Ciências Exatas de São José do Rio Preto, SP, Brasil. Master's thesis. Orienting Professor: Maria Tercília Vilela de Azeredo Oliveira.
Malpighian tubule cell nuclei of the triatomid Rhodnius prolixus were studied under stress conditions provoked by starvation and by temperature shocks (hypo- and hyperthermic). Starvation most drastically affected both males and females after 21 days.
Staining was done with lacto-acetic orcein, which stains proteins complexed with DNA (acidophilic reaction), and impregnation by silver ions, responsible for the staining of proteins present in nucleolar bodies, a nucleolina (C23) and numatrina (B23) (acidophilic reactions). The alterations most commonly observed with starvation involved changes in chromatin distribution, increased nuclear and nucleolar areas, nuclei with irregular outlines and nucleolar fusion.
Extenuation of the chromatin was determined by the method of critical electrolyte concentrations (CEC), which uses a solution of toluidine blue with MacIlvaine buffer and different concentrations of magnesium cloride. With continued starvation the CEC point decreases, which indicates a decrease in chromatin. Liberation of PO42- groups then occurs and these bind to inorganic cations in the form of magnesium chloride. The nucleus then become greenish, losing the metachromasy (violet blue).
It is possible to confirm the fusion of nucleolar corpuscles during starvation by the variant of CEC, which was observed by silver impregnation. Insects which were fed after 30 days of starvation showed only a slight recuperation of chromatin condensation. An increase in the concentration, in moles, of magnesium chloride was observed with the CEC method. The nucleolar corpuscles were unfused.
Silver impregnation and CEC techniques were used for the temperature shock studies. In this type of stress the same alterations were observed as in starvation stress (chromatin extenuation, nucleolar fusion and polyploidy). Occurrence varied with the type of shock and the time of exposition.
Nuclei were altered more drastically with a hyperthermic shock (40oC) than with a hypothermic shock. However, with longer periods of exposition (12 h) to the shocks, both at high and low temperatures, there was a greater extenuation of the chromatin, which ended the metachromasy of the protein-DNA complexes, with a lowered concentration of magnesium chloride.
Nuclear metabolic mechanisms which serve to protect the cell were observed in the two types of stress studied. These mechanisms involve an increase in nuclear and nucleolar areas and chromatin extenuation, which reflect an increased synthesis of proteins as a consequence of these stress factors.
Publication supported by FAPESP.
Risk calculation in genetically heterogeneous diseases: development of the method and application to congenital deafness
(Cálculo de riscos em doenças geneticamente heterogêneas: desenvolvimento de método e aplicação no caso da surdez congênita)
Maria Cristina Célia Braga*
*1998. Departamento de Biologia, Instituto de Biociências, Universidade de São Paulo, São Paulo, SP, Brasil. Master's thesis. Orienting Professor: Paulo A. Otto.
To estimate the recurrence risks in cases of genetic counseling of nonsyndromic deafness, a heterogeneous condition that may be due not only to some hereditary monogenic mechanism but also to several environmental factors, a survey of this defect was performed among families from three different specialized institutions in the city of São Paulo.
Using our own data and similar reliable data published in the Brazilian literature, we found that 84% of the cases in Brazil were of environmental origen. This figure is much higher than in developed countries, the statistics of which is used traditionally in the calculation of composite risks for the genetic counseling of nonsyndromic deafness.
The relative proportions of the hereditary variants of this defect, do not vary significantly in magnitude from one geographic region to another. Combining our own data with similar estimates from the literature, we obtained estimates of 78, 19 and 3% for autosomal recessive, autosomal dominant and X-linked recessive cases, respectively.
Using these estimated proportions, including (A) or not (B) environmental factors, we calculated the recurrence risks for most situations found in the genetic counseling of deafness.
|Non-affected non-consanguineous couple, one affected child||0.04||0.22|
|Non-affected first degree cousins, one affected child||0.17||0.25|
|Non-affected couple, two affected children||-||0.25|
|Non-consanguineous couple, one parent affected||0.01||0.08|
|One spouse affected, one affected child||-||0.40|
|First degree cousins, one parent affected, one affected child||-||0.46|
|Non-consanguineous couple, both parents affected||0.03||0.17|
|First degree cousins, both parents affected||0.06||0.37|
|Non-consanguineous couple, both parents affected, one affected child||0.51||0.74|
|First degree cousins, both parents affected, one affected child||0.45||0.53|
For the calculation of the risks shown above we used an average number of loci for non-syndromic autosomal recessive deafness of 30, which corresponds to an average gene frequency of 0.004. At this frequency, the relative risk of deafness for children born to first degree cousins turns out to be about 16 times higher than in the offspring of unrelated parents.
Accounts for different frequencies of environmentally provoked deafness was also developed.
A detailed study was made of a large family with more than 30 individuals affected by an incompletely penetrant autosomal dominant type deafness. This form had a late age of onset and a progressive pattern in this family. Global and age-specific penetrance values were estimated from the data and used in the calculation of recurrence risks for all its members.
The main practical conclusions of our study were:
a) In cases where environmental factors cannot be excluded as a causal determinant of the defect, the risks traditionally found in the international specialized literature cannot be directly applied to our population, due to the high local prevalence rates of environmental cases.
b) Parental consanguinity distorts the probabilities, even when environmental causes are properly discarded. When there is no parental consanguinity, the population prevalences can be used in the composite risks without any correction.
c) If environmental causes are properly discarded, the recurrence risk of deafness in the next sib is about 25%, even thoug the parental consanguinity almost exclusively indicates recessive inheritance. If environmental causation cannot be excluded, on the other hand, the occurrence of an affected isolated case in the offspring of first cousins indicates a recessive inheritance with high probability, the same not being true when there is no parental consanguinity.
d) Marriages taking place between two affected individuals is common in cases of deafness, though it had received no special attention until now. When this happens, the risks of affected offspring range from 3 to 18% when the couples are unrelated, and from 6 to 37% when they are first degree cousins, whether we take or not into account environmental factors. When an affected child already exists, the risks range from 51 to 74% and from 45 to 53%, respectively.
e) Recurrence risks for all situations are similar independent of whether the index cases are male or female, due to the low relative proportion of X-linked cases among all hereditary cases of the defect.
Research supported by CNPq.
Publication supported by FAPESP.
Floral biology and isoenzymatic variability in Ocimum selloi Benth
(Biologia floral e variabilidade isoenzimática em Ocimum selloi Benth)
Cláudio Lúcio Fernandes Amaral*
*1997. Departamento de Fitotecnia (Genética e Melhoramento), Universidade Federal de Viçosa, Viçosa, Minas Gerais, Brasil. Master's thesis. Orienting Professor: Vicente Wagner Dias Casali.
The floral biology and the reproductive mechanisms as well as the inter- and intrapopulational isoenzymatic variability of Ocimum selloi Benth was studied. Floral activity was found to have three phases: in the first one, pre-anthesis, pollination occurs; in the second one, anthesis, floral bud opening and subsequently the asychronic stamen externalization occur, and in the third phase, post-anthesis, ovule fecundation occurs. Since the flower was closed at pollination time, it was considered to be cleistogamic. Fecundation occurred in the post-anthesis phase, when the flower was already opened. Thus, low rates of cross fertilization are possible, as a flower from one plant may receive pollen from another plant by means of pollinating insects. Even if cleistogamy does not occur, the ovules are still effectively fertilized because in the post-anthesis phase the stigma still remains receptive to cross fertilization. The enzymatic systems alcohol dehydrogenase, esterase, acid phosphatase, glutamate dehydrogenase, glutamate oxaloacetate transaminase, isocitrate dehydrogenase, leucine aminopeptidase, malate dehydrogenase, peroxidase and shikmic dehydrogenase, were tested. They were adequate for identifying, characterizing and differentiating populations and their respective individuals.
Cytogenetic studies of fish (Pimelodidae and Rhamdiidae) from the Tibagi River basin/Paraná
(Estudos citogenéticos em peixes das famílias Pimelodidae e Rhamdiidae da bacia do rio Tibagi/PR)
Ana Cláudia Swarça*
*1998. Pós-graduação em Genética e Melhoramento, Departamento de Biologia Geral, Universidade Estadual de Londrina, Londrina, PR, Brasil. Master's thesis. Orienting Professor: Ana Lúcia Dias.
Cytogenetic studies were made of Pimelodidae (Pimelodus maculatus, Pinirampus pirinampu) and Rhamdiidae (Pimelodella sp., Pimelodella aff. gracilis) from the Tibagi River basin in the State of Paraná. P. maculatus had 2n = 56 chromosomes (20m + 20sm + 10st + 6a), P. pirinampu had 2n = 50 (26m + 12sm + 2st + 10a), Pimelodella sp. had 2n = 46 (34m + 12sm) and P. aff. gracilis 2n = 52 (24m + 18sm + 4st + 6a). Nucleolus organizer regions (NOR) were observed in a pair of chromosomes in the telomeric region through impregnation by AgNO3 in all specimens. In Pimelodus maculatus an NOR was located in the long arm of a pair of subtelocentric chromosome. In P. pirinampu, Pimelodella sp. and P. aff. gracilis an NOR was located in the short arm of a pair of subtelocentric, metacentric and submetacentric chromosomes, respectively. In P. pirinampu size variations in the NOR among the paired chromosomes were observed, probably due to genic amplification in this region. Staining through chromomycin A3 showed the NOR regions in all specimens. In P. pirinampu, besides the nucleolar chromosome pair, fluorescent markings were observed in the telomeric and centromeric regions of other chromosomes which seemed to correspond to the distribution patterns of the constitutive heterochromatin. In this species several chromosomes with strongly heterochromatic telomeric and centromeric regions were observed with C banding. The constitutive heterochromatin in P. maculatus was weakly visualized in telomeric and/or centromeric regions of several chromosomes. A restriction enzyme AluI was used in these two specimens of pimelodideos and the pattern of reaction observed corresponded to the heterochromatin constitutive distribution. Pimelodella sp. and P. aff. gracilis had a small quantity of heterochromatin in the majority of their chromosomes, but in these two specimens a strongly heterochromatic chromosome pair was observed in the telomeric region mainly in P. aff. gracilis.
Research supported by CAPES.
BiP-storage protein interactions and isolation of BiP cDNA and BiP genomic clones from soybean [Glycine max (L.) Merrill]
(Determinação da interação BiP ("binding protein")-proteína de reserva e isolamento de clones de cDNA e genômicos de BiP da soja [Glycine max (L.) Merrill])
José Edson Fontes Figueiredo*
*1998. Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brasil. Doctoral thesis. Orienting Professors: Dr. Marcos Luiz dos Mares-Guia and Dra. Elizabeth Pacheco Batista Fontes.
The endoplasmic reticulum (ER) luminal binding protein (BiP) is thought to be a key mediator of folding and assembly of de novo synthesized secretory proteins. A maize (Zea mays L.) BiP antibody was used to identify its homolog from soybean [Glycine max (L.) Merril]. BiP accumulation in developing soybean seeds seems to be coordinated with the onset of active storage protein synthesis. A co-immunoprecipitation assay was used to detect soybean BiP:b-conglycinin interactions. Either a maize BiP antibody or a b-conglycinin antibody co-immunoprecipitated with the reciprocal protein from the whole seed protein extract enzymatically depleted of adenosine 5'-triphosphate (ATP), while an unrelated antibody failed to immunoprecipitate either one. BiP:b-conglycinin complexes were completely dissociated by ATP addition, a diagnostic feature of molecular chaperone-mediated interaction. However, a very small fraction of b-conglycinin was found to be associated with BiP in the whole cell protein extracts from immature seeds. These results indicate a transient association between BiP and b-conglycinin subunits and suggest its involvement in the biosynthetic transport pathway of storage proteins to protein bodies.
A BiP cDNA clone (cUFVBiP1) was isolated from a seed expression library and molecular analysis of the soybean BiP gene family was performed. cUFVBiP1 encodes a pre-protein of 668-amino acid residues and Mr 85.729 kDa. After processing, a mature protein of 641 amino acids (Mr 70,790) and isoelectric point 5.08 displayed several features shared by Hsp70 proteins and specifically by BiP protein: the ATP binding domain (sequence LGIETVGGV), the conserved peptide TVIGIDLGTTYSC found in all members of the Hsp70 family which are stress induced, a putative calmodulin-binding domain (NRALGKLRREAERAKRALSSQ), and the hydrophobic ER N-terminus signal peptide and the tetrapeptide HDEL at the C-terminus, which is responsible for the ER retention and retrieval of the protein. Amino acid comparison with the encoded products of other previously sequenced soybean BiP cDNAs (sBiPA, sBiPB, sBiPC) (Kalinski et al., 1995) showed 86, 92 and 96% identity, respectively. At the nucleotide level, the high conservation of the soybean BiP genes was lower within the 5' and 3' untranslated regions. While the 5' and 3' untranslated regions of sBiPA, sBiPB, and sBiPC were quite dissimilar, these regions were more conserved between sBiPB and cUFVBiP1.
In order to gain more insight into the complexity of the soybean BiP gene family, an extensive DNA gel blot analysis of Doko and IAC 100 varieties was performed using restriction endonucleases-digested leaf DNA and the cUFVBiP1 clone as a probe. A very similar hybridization pattern for all restriction enzymes was observed between these two varieties. Several high molecular bands were observed even when restriction endonucleases that do not digest cUFVBiP1 were used. Some of these bands probably are the result of hybridization between clone cUFVBiP1 with other members of the highly conserved hsp70 gene family.
The cUFVBiP1 clone was used to screen a lambda EMBL3 genomic library constructed with leaf DNA from soybean cv Roonocke. Two clones were isolated and sequenced. The nucleotide sequence identity between these clones was very high (>98%) and shared the same structural organization, with intron and exons displaying the same size and localization. Moreover, these two clones also showed high identity with the genomic spinach BiP gene at the nucleotide sequence level (89%) and the structural organization (introns and exons).
Beta-galactosidase activity and characterization of geneticin-resistant Kluyveromyces lactis mutants
(Atividade de beta-galactosidase e caracterização de mutantes de Kluyveromyces lactis resistentes à geneticina)
Eriana Serpa Barreto*
*1998. Pós-graduação em Microbiologia Agrícola, DMB, CCB, Universidade Federal de Viçosa (UFV), Viçosa, MG, Brasil. Master's thesis. Orienting Professor: Flávia Maria Lopes Passos.
Geneticin-resistant Kluyveromyces lactis mutants were isolated and characterized with the purpose of isolating a b-galactosidase-secreting strain. The mutagenic agent used was ultraviolet light and geneticin resistance was tested over a concentration range of 0.5 to 1.0 mg/ml. Seven-minute radiation resulted in 1 to 5% survival of the population. Five thousand geneticin-resistant mutant colonies were tested. None of these showed b-galactosidase extracellular activity. However, one mutant resistant to 1.0 mg/ml geneticin presented a high amount of extracellular protein, 1.75 times more than the wild type K. lactis. No differences among mutants and wild type cells were observed in the assimilation of six carbon sources: sucrose, lactose, galactose, mannitol, raffinose and cellobiose. Resistance to geneticin did not affect the growth kinectics of K. lactis in ultrafiltered cheese whey and YNB (yeast nitrogen base without amino acids) with or without lactose and sucrose as additional carbon sources. Mutants showed growth rates and maximum population similar to the wild type cell in the culture medium tested. A b-galactosidase production peak occurred at around 12 h of growth, during the exponential phase. Karyotype analysis showed differences in chromosomic band patterns between mutants and wild type K. lactis.