Scielo RSS <![CDATA[Genetics and Molecular Biology]]> vol. 25 num. 1 lang. en <![CDATA[SciELO Logo]]> <![CDATA[<B>Editorial</B>]]> <![CDATA[<B>Fragile X founder effect and distribution of CGG repeats among the mentally retarded population of Andalusia, South Spain</B>]]> Fragile X syndrome is the most common inherited form of mental retardation. We investigated the prevalence of the Fragile X syndrome in the population with mental retardation of unknown etiology in Andalusia, South Spain. We analyzed 322 unrelated patients (280 males and 42 females), and found a fragile X syndrome frequency of 6.5%. Among the non-fragile X chromosomes, the 29 CGG repeat was the most common allele. At the linked microsatellite DXS548 locus, we found a new allele which we called "allele 10" (17 CA). Similar to other south European populations, allele 2 (25 CA) at the DXS548 locus and the fragile X allele were in linkage disequilibrium supporting the idea of a common founder chromosome predisposing to the CGG expansion. <![CDATA[<B>Diagnosis of patients with Prader-Willi and Angelman Syndromes</B>: <B>the importance of an overall investigation</B>]]> Seventy-two patients with clinical diagnoses of Prader-Willi (PWS; n = 28 patients) or Angelman syndromes (AS; n = 44 patients) were submitted to chromosome analysis, SNRPN-SNURF exon 1 methylation assay, and microsatellite genotyping. Analysis of the methylation pattern confirmed the PWS diagnosis in 18 out of 28 patients and the AS diagnosis in 20 out of 44 patients. FISH and microsatellite analysis detected a deletion in 30 patients (14 PWS and 16 AS). Eight patients had normal FISH results (4 PWS and 4 AS); microsatellite markers showed that these patients had a uniparental disomy (UPD). Based on this study, we propose a strategy for the routine diagnosis of these syndromes that consists of the following steps: 1) methylation analysis, which does not require parental samples; 2) microsatellite genotyping of patient and parents to differentiate deletions, UPD and imprinting mutations; and 3) FISH for otherwise uninformative cases, and whenever parental samples are not available. Of the 34 patients whose PWS or AS diagnoses were not confirmed by laboratory tests, five presented a small extra marker chromosome, identified in three of them as an inv dup(15). One AS patient carried a balanced t(15;15) translocation associated with paternal UPD. Therefore G-banded chromosome analysis should be performed on all such patients, to detect possible structural rearrangements. <![CDATA[<B>Identification and characterization of polymorphisms at the HSA <FONT FACE=Symbol>a</FONT><SUB>1</SUB><I>-acid glycoprotein</I> (<I>ORM*</I>) gene locus in Caucasians</B>]]> Human alpha1-acid glycoprotein (AGP) or orosomucoid (ORM) is a major acute phase protein that is thought to play a crucial role in maintaining homeostasis. Human AGP is the product of a cluster of at least two adjacent genes located on HSA chromosome 9. Using a range of restriction endonucleases we have investigated DNA variation at the locus encoding the AGP genes in a panel of healthy Caucasians. Polymorphisms were identified using BamHI, EcoRI, BglII, PvuII, HindIII, TaqI and MspI. Non-random associations were found between the BamHI, EcoRI, BglII RFLPs. The RFLPs detected with PvuII, TaqI and MspI were all located in exon 6 of both AGP genes. The duplication of an AGP gene was observed in 11% of the indiviuals studied and was in linkage disequilibrium with the TaqI RFLP. The identification and characterization of these polymorphisms will prove useful for other population and forensic studies. <![CDATA[<B>Beta-globin gene cluster haplotypes in Venezuelan sickle cell patients from the State of Aragua</B>]]> Seven polymorphic sites in the beta-globin gene cluster were analyzed on a sample of 96 chromosomes of Venezuelan sickle cell patients from the State of Aragua. The Benin haplotype was predominant with a frequency of 0.479, followed by the Bantu haplotype (0.406); a minority of cases with other haplotypes was also identified: atypical Bantu A2 (0.042), Senegal (0.031), atypical Bantu A7 (0.021) and Saudi Arabia/Indian (0.021) haplotypes; however, the Cameroon haplotype was not identified in this study. Our results are in agreement with the historical records that establish Sudanese and Bantu origins for the African slaves brought into Venezuela. <![CDATA[<B>Mitochondrial variability in the D-loop of four equine breeds shown by PCR-SSCP analysis</B>]]> A fragment of 466 base pairs from a highly variable peripheral region of the mitochondrial D-loop of horses was amplified and analyzed by single stranded conformational polymorphism (SSCP). Fourteen distinct SSCP variants were detected in 100 horses belonging to four breeds (Arabian, ARB; Thoroughbred, TB; Argentinian Creole, ARC; and Peruvian Paso from Argentina, PPA). Each breed showed four to eight SSCP variants, many of which were shared between two or three of the studied breeds. Arabian horses were the most variable (eigth variants), with three variants unique to the breed. PPA and ARC showed two and one characteristic SSCP variants, respectively, while TB shared all its variants with at least one of the other breeds. An analysis based on the presence/absence of the variants revealed a closer relationship between PPA and TB, which was not completely unexpected considering the mixed ancestry of the PPA mares. The results also confirm the efficiency of SSCP to detect variability in horse mitochondrial DNA. <![CDATA[<B>Influence of obesity gene in quantitative traits of swine</B>]]> Genotype data of 477 animals of several swine races (Landrace - LD, Large White - LW, Pietrain - PI, LWXLDXPI, Piau, Monteiro, and unknown race) were obtained to determine the allele frequency of the obesity gene. Genotype data of 174 crossbred swine (LWXLDXPI) were also obtained, in order to assess its correlation with carcass evaluation data (lean meat percentage, backfat thickness at P2, loin eye area, adjacent fat area, total fat and meat). Finally, genotype data of 96 pure swine (Landrace, Large White and Pietrain) were collected, to establish its relation with meat quality (drip loss, meat color, texture analysis and intramuscular fat) and carcass evaluation data (lean meat percentage; ham, loin, shoulder and belly weights; and backfat thickness at P2). This work also aimed associating EPDs (expected progeny differences) for litter size, daily weight gain and backfat thickness with genotype data of 49 Large White males and 54 Landrace females. Genotyping was done on animal blood by PCR-RFLP, based on Stratil et al. (1997). Statistical analysis was done by using SAS software for variance analysis between genotypes and data for each cited class. For purebred animals, a mixed model was used, with sire within race as random effect. The allelic frequencies of alleles T and C were, respectively: 0.8142; 0.1857 (Landrace); 0.9125; 0.0875 (Large White); 0.9433; 0.0566 (Pietrain); 0.8333; 0.1666 (LWXLDXPI); 0.2500; 0.7500 (Piau); 0.8750; 0.1250 (Monteiro), and 0.8870; 0.1130 (unknown race). Since the highest allele C frequency occurred in Piau, we suggest that this allele could be associated with fat accumulation. In the Landrace race, a study was done separating the frequencies of 2 generations (great-grandfather and grandfather), and the differences confirmed by a Chi-square test, a higher frequency of allele C having been found in the grandparental generation. This suggests that this allele could be eliminated by selection from the great-grandparental generation, when the male grandparent is replaced by the great-grandparental generation. The obesity gene did not influence any of the carcass evaluation data from crossbred animals. In pure swine, where the only genotypes were TT and TC, it greatly influenced shoulder weight and meat texture, with the highest average in heterozygotes (shoulder: 4.07 vs. 3.93; texture: 2.62 vs. 1.82), suggesting better carcass quality and worse meat quality than in homozygotes. The obesity gene did not influence any trait in the expected progeny difference (EPD) study. <![CDATA[<B>Evaluation of polymorphism in ten microsatellite loci in Uruguayan sheep flocks</B>]]> The allele frequencies of 10 microsatellite loci previously described for sheep as BM1314, BM6526, OarFCB128, OarHH64, OarCP20, OarHH47, OarFCB48, OarHH35, OarHH72 and BM2508 were estimated for the Uruguayan flocks. A representative sample of 101 individuals composed by the two predominant breeds (76% Corriedale and 24% Australian Merino) was used. The sample did not show a significant tendency towards substructuring, in spite of presenting some significantly different allele frequencies between races. The Corriedale sample presents three loci in which the presence of null alleles is possible. The markers were highly variable, showing between 7 and 15 alleles each. The Polymorphism Information Content Index ranged from 0.63 to 0.87 and the Exclusion Probability from 0.39 to 0.75 for a cumulative Exclusion Probability of 99.98%. These results suggest the effectiveness of this set of loci for testing genetic relatedness. This is the first report of microsatellite variation in Corriedale. <![CDATA[<B>Three synonymous genes encode calmodulin in a reptile, the Japanese tortoise, <I>Clemmys japonica</B></I>]]> Three distinct calmodulin (CaM)-encoding cDNAs were isolated from a reptile, the Japanese tortoise (Clemmys japonica), based on degenerative primer PCR. Because of synonymous codon usages, the deduced amino acid (aa) sequences were exactly the same in all three genes and identical to the aa sequence of vertebrate CaM. The three cDNAs, referred to as CaM-A, -B, and -C, seemed to belong to the same type as CaMI, CaMII, and CaMIII, respectively, based on their sequence identity with those of the mammalian cDNAs and the glutamate codon biases. Northern blot analysis detected CaM-A and -B as bands corresponding to 1.8 kb, with the most abundant levels in the brain and testis, while CaM-C was detected most abundantly in the brain as bands of 1.4 and 2.0 kb. Our results indicate that, in the tortoise, CaM protein is encoded by at least three non-allelic genes, and that the ‘multigene-one protein' principle of CaM synthesis is applicable to all classes of vertebrates, from fishes to mammals. <![CDATA[<B>Nuclear DNA content determination in Characiformes fish (Teleostei, Ostariophysi) from the Neotropical region</B>]]> In the present study, nuclear DNA content was analyzed in 53 species of Characiformes fish from the Neotropical region. Diploid number ranged from 2n = 48 in Astyanax fasciatus, Gymnocorymbus ternetzi and Hyphessobrycon griemi to 2n = 102 in Potamorhina squamoralevis, with a modal number of 54 chromosomes. Nuclear DNA content ranged from 1.70 ± 0.04 pg of DNA per diploid nucleus in Acestrorhynchus pantaneiro to 3.94 ± 0.09 pg in Tetragonopterus chalceus. A general analysis showed a mean value of 2.9 pg of DNA per diploid nucleus. Very similar DNA content values were observed in the species of the family Cynodontidae which showed a variation of 3% between the two genera studied. Small variations were observed between populations of Gymnocorymbus ternetzi, Astyanax fasciatus and Moenkhausia sanctaefilomenae (Characidae, Tetragonopterinae). The subfamilies Tetragonopterinae and Acestrorhynchinae (Characidae) presented the widest range, about 96%. Even in those families in which diploid number and karyotypic formulae were conserved such as the families Anostomidae, Curimatidae, and Prochilodontidae, episodes leading to losses or gains of genetic material became fixed in their evolutionary history. <![CDATA[<B>Evaluation of the defensive behavior of two honeybee ecotypes using a laboratory test</B>]]> Honeybee defensive behavior is a useful selection criterion, especially in areas with Africanized honeybees (Apis mellifera L). In all genetic improvement programs the selected characters must be measured with precision, and because of this we evaluated a metabolic method for testing honeybee defensive behavior in the laboratory for its usefulness in distinguishing between honeybee ecotypes and selecting honeybees based on their level of defensive responses. Ten honeybee colonies were used, five having been produced by feral queens from a subtropical region supposedly colonized by Africanized honeybees and five by queens from a temperate region apparently colonized by European honeybees. We evaluate honeybee defensive behavior using a metabolic test based on oxygen consumption after stimulation with an alarm pheromone, measuring the time to the first response, time to maximum oxygen consumption, duration of activity, oxygen consumption at first response, maximum oxygen consumption and total oxygen consumption, colonies being ranked according to the values obtained for each variable. Significant (p < 0.05) differences were detected between ecotypes for each variable but for all variables the highest rankings were obtained for colonies of subtropical origin, which had faster and more intense responses. All variables were highly associated (p < 0.05). Total oxygen consumption was the best indicator of metabolic activity for defensive behavior because it combined oxygen consumption and the length of the response. This laboratory method may be useful for evaluating the defensive behavior of honey bees in genetic programs designed to select less defensive bees. <![CDATA[<B>Esterase-3 polymorphism in the sugarcane borer <I>Diatraea saccharalis</I> (Lepidoptera, Pyralidae)</B>]]> The migration rate of esterases and their substrate specificity for 4-methylumbelliferyl esters (acetate, propionate, and butyrate) and alpha- and beta-naphthyl esters were analyzed in Diatraea saccharalis by starch gel electrophoresis. Substrate preference of esterases was observed with Est-2 and Est-8 isozymes showing substrate specificity for 4-methylumbelliferyl esters and Est-4 isozyme showing specificity for 4-methylumbelliferyl butyrate and alpha-naphthyl butyrate. Allele variation was detected at the Est-3 locus. Two alleles, Est-3F and Est-3S, were identified in pupae with fluorogenic and ester-naphthyl substrates. Chi-square analysis showed no differences between the observed genotypic frequencies and those expected on the basis of Hardy-Weinberg frequencies for the Est-3 locus (chi² = 2.4; p < 0.01). The negative value for the Wright's fixation index (F = -0.2096) calculated for the D. saccharalis population maintained under laboratory conditions indicates an excess of heterozygotes, however, the observed Hardy-Weinberg equilibrium indicates that in the laboratory the population of D. saccharalis behaved as if the moth were randomly mating in nature. The high level of heterozygosity at the Est-3 locus indicates also that this esterase may be a good genetic marker for studies of natural D. saccharalis populations. <![CDATA[<B>Variability among inbred lines and RFLP mapping of sunflower isozymes</B>]]> Eight isozyme systems were used in this study: acid phosphatase (ACP), alcohol dehydrogenase (ADH), esterase (EST), glutamate dehydrogenase (GDH), malate dehydrogenase (MDH), phosphoglucoisomerase (PGI), 6-phosphogluconate dehydrogenase (PGD), and phosphoglucomutase (PGM). The polymorphism of these enzyme systems was studied in 25 elite inbred lines. A total of 19 loci were identified, but only eight of them were polymorphic in the germplasm tested. The polymorphic index for the eight informative markers ranged from 0.08 to 0.57, with a mean value of 0.36. Five isozyme loci were mapped in F2:3 populations with existing RFLP data. Est-1, Gdh-2 and Pgi-2 were mapped to linkage groups 3, 14 and 9, respectively. As in previous reports, an ACP locus and a PGD locus were found to be linked, both located in linkage group 2 of the public sunflower map. <![CDATA[<B>Factors influencing electroporation-mediated gene transfer to <I>Stylosanthes guianensis</I> (Aubl.) Sw. protoplasts</B>]]> In order to develop a high-efficiency and reproducible transformation protocol for Stylosanthes guianensis we assessed the biological and physical parameters affecting plant electroporation protoplasts. Energy input, as combinations of electric field strengths discharged by different capacitors, electroporation buffer and DNA form were evaluated. Transformation efficiency was assayed in vivo as transient reporter gene expression, using the GFP-coding gene mgfp5 driven by a CaMV 35S constitutive promoter. Energy input and electric field strength had a critical influence on transgene expression with higher transformation levels being achieved with 250 discharged by 900 and 1000 muF capacitors. Linear plasmid DNA, the absence of chloride and the presence of calcium ions also increased transient gene expression, albeit not significantly. <![CDATA[<B>Acid phosphatase polymorphism within and among populations of Cauliflower (<I>Brassica oleracea</I> var<I> botrytis</I>)</B>]]> Eighty-one lines of cauliflower (Brassica oleracea var. botrytis) from 12 populations used to produce commercial hybrids in Brazil were screened for polymorphism in the acid phosphatase system, in order to evaluate the usefulness of this marker for the determination of the parental contamination level in hybrid seeds. Little polymorphism was detected in the examined lines, but the system appeared to be very useful for hybrid identification, since the only condition required was polymorphism between the two parental lines. If the analyzed lines were used for hybrid production, 8.4% and 12.3% of the possible crosses would result in hybrids which can be positively identified using the APS-1 and B1 loci, respectively. If only one plant of each homozygous type (SS or FF) was analyzed in each population, 41% and 50% of the possible crosses would result in hybrids which can be positively identified using the APS-1 and B1 loci, respectively. <![CDATA[<B>Effects of <I>Maytenus ilicifolia</I> Mart. and <I>Bauhinia candicans</I> Benth infusions on onion root-tip and rat bone-marrow cells</B>]]> Medicinal plants are widely used to treat various diseases, and in Brazil the plants Maytenus ilicifolia Mart. and Bauhinia candicans Benth are commonly used in popular medicine. However, there are a large number of compounds in plants which can produce alterations in genetic material, and this study was conducted to investigate any possible mutagenic and cytotoxic effects that M. ilicifolia and B. candicans infusions may have on the cell cycle and chromosomes. Infusions were prepared with in natura leaves to give two concentrations of infusions, one at the concentration normally used by the population in general and the other at 10 times this value (i.e. 3.5 and 35 mg/mL for M. ilicifolia and 0.465 and 4.65 mg/mL for B. candicans). Onion (Allium cepa L.) root-tip cells (RTC) and Wistar rat bone-marrow cells (BMC) were used as test systems in in vivo assays. The M. ilicifolia infusions at both concentrations, and the B. candicans infusion at the lower concentration, had no statistically significant depressive mitotic effect on RTC. A statistically significant depressive mitotic effect on RTC was found with the more concentrated (4.65 mg/mL) B. candicans infusion as compared with a negative control. In BMC, infusions of B. candicans and M. ilicifolia produced no statistically significant increase in the number of chromosome alterations or rates of cell division as compared to controls. The significance of these findings are discussed in the light of the use of these plants as therapeutic agents. <![CDATA[<B>Genetic instability of sugarcane plants derived from meristem cultures</B>]]> The RADP (Random amplified polymorphic DNA) technique was used to detect tissue-culture-induced variations in sugarcane. Plants of the Brazilian variety RB83-5486 propagated via rhizomes and via meristem cultures were studied. The polymorphism rate for 98 RAPD loci was 6.93% when the plants derived from meristems. Besides, in order to evaluate the influence of the number of subcultures on the generation of somaclonal variation, field-grown RB83-5486 plants derived from 10 meristems were studied after five subcultivations. Although different rates of polymorphism were observed, there was no direct association with the stage of subcultivation. The analysis of plants of two sugarcane varieties cultivated in vitro from meristems showed that variety RB83-5486 was more unstable than variety SP80-185. <![CDATA[<B>Construction and expression of aspartic protease from <I>Onchocerca volvulus</I>* as ompA fusion protein in a mutant strain of <I>Salmonella typhimurium</B></I>]]> Two constructions in pHS164 vector were designed to permit expression of OV7A and OV4A inserts encoding the N-terminal and C-terminal portion of an aspartic protease from Onchocerca volvulus, respectively. A novel 39 kD protein ompA-OV7A fusion protein was stably expressed as ompA fusion in a modified strain of Salmonella typhimurium strain SL5000 and E.coli strain JM109. Expression of the fusion protein in bacterial strains harboring the constructs were evaluated by western blotting. E.coli and Salmonella lysates were fractionated by 10% SDS-PAGE gel and then immobilized to nitrocellulose membrane by electroblotting. Primary polyclonal antibody generated in rats against the GST-OV7A fusion protein was used in the Western blots. It remains to be seen whether the fusion protein expressed in vivo will promote effective immune response. <![CDATA[<B>Differentiation of mouse embryonic stem cells and their hybrids during embryoid body formation</B>]]> We studied the karyotypes of three hybrid clones of mouse embryonic stem cells and murine splenocytes (two having near diploid and one having near tetraploid chromosome numbers) and the characteristics of their differentiation during the formation of embryoid bodies. The X chromosome originating from embryonic stem cells may be lost in hybrids with a near diploid chromosome number and reprogramming of the "somatic" X may occur. The morphological data we obtained using light and electron microscopy revealed a correlation between the karyotype constitution of hybrid cells and their differentiation during the formation of embryoid bodies. At the beginning of development, the embryoid bodies derived from hybrid cells already showed an advanced degree of differentiation. The production of significant quantities of cartilage was typical for hybrid cells with near tetraploid chromosome numbers. The hybrid cells showed restricted pluripotent capacity and were already committed when they started to differentiate into embryoid bodies.