Sergio Russo Matioli

Molecular adaptation of Drosophila melanogaster lysozymes to a digestive function.

A lysozyme (pI 5.5) was purified to homogeneity from heated acid extracts of Drosophila melanogaster larvae, using gel filtration in a Superose column and ion-exchange chromatography in a Mono Q column. The final yield was 67%. The purified lysozyme with Mr 13,700 (determined by SDS-polyacrylamide gel electrophoresis) decreases in activity and has its pH optimum displaced towards acidic values and Km increases as the ionic strength of the medium becomes higher. The lysozyme is resistant to a cathepsin D-like proteinase present in cyclorrhaphous Diptera and displays a chitinase activity which is 11-fold higher than that of chicken lysozyme. Microsequencing of an internal peptide of the purified lysozyme showed that this enzyme is the product of the previously sequenced Lys D gene. The results suggest that the product of the Lys P gene has pI 7.2, a pH optimum around 5 and is not a true digestive enzyme. The most remarkable sequence convergence of D. melanogaster lysozyme D and lysozymes from vertebrate foregut fermenters are serine 104 and a decrease in the number of basic amino acids, suggesting that these features are necessary for digestive function in an acid environment. Adaptive residues putatively conferring stability in an acid proteolytic environment differ between insects and vertebrates, probably because they depend on the overall three-dimensional structure of the lysozymes. A maximum likelihood phylogeny and inferences from insect lysozyme sequences showed that the recruitment of lysozymes as digestive enzymes is an ancestral condition of the flies (Diptera: Cyclorrhapha).

Clinical variability in calpainopathy: What makes the difference?

Limb girdle muscular dystrophies (LGMD) are a heterogeneous group of genetic disorders characterised by progressive weakness of the pelvic and shoulder girdle muscles and a great variability in clinical course. LGMD2A, the most prevalent form of LGMD, is caused by mutations in the calpain-3 gene (CAPN-3). More than 100 pathogenic mutations have been identified to date, however few genotype : phenotype correlation studies, including both DNA and protein analysis, have been reported. In this study we screened 26 unrelated LGMD2A Brazilian families (75 patients) through Single-Stranded Conformation Polymorphism (SSCP), Denaturing high-performance liquid chromatography (DHPLC) and sequencing of abnormal fragments which allowed the identification of 47 mutated alleles (approximately 90%). We identified two recurrent mutations (R110X and 2362-2363AG>TCATCT) and seven novel pathogenic mutations. Interestingly, 41 of the identified mutations (approximately 80%) were concentrated in only 6 exons (1, 2, 4, 5, 11 and 22), which has important implications for diagnostic purposes. Protein analysis, performed in 28 patients from 25 unrelated families showed that with exception of one patient (with normal/slight borderline reduction of calpain) all others had total or partial calpain deficiency. The effects of type of mutation, amount of calpain in the muscle, gender and ethnicity of affected patients on clinical course (age of onset and ascertainment) were analysed. Interestingly, it was observed that, on average, African–Brazilian calpainopathy patients are more severely affected than Caucasians.

Epistasis affecting litter size in mice

Litter size is an important reproductive trait as it makes a major contribution to fitness. Generally, traits closely related to fitness show low heritability perhaps because of the corrosive effects of directional natural selection on the additive genetic variance. Nonetheless, low heritability does not imply, necessarily, a complete absence of genetic variation because genetic interactions (epistasis and dominance) contribute to variation in traits displaying strong heterosis in crosses, such as litter size. In our study, we investigated the genetic architecture of litter size in 166 females from an F2 intercross of the SM/J and LG/J inbred mouse strains. Litter size had a low heritability (h2 = 12%) and a low repeatability (r = 33%). Using interval‐mapping methods, we located two quantitative trait loci (QTL) affecting litter size at locations D7Mit21 + 0 cM and D12Mit6 + 8 cM, on chromosomes 7 and 12 respectively. These QTL accounted for 12.6% of the variance in litter size. In a two‐way genome‐wide epistasis scan we found eight QTL interacting epistatically involving chromosomes 2, 4, 5, 11, 14, 15 and 18. Taken together, the QTL and their interactions explain nearly 49% (39.5% adjusted multiple r2) of the phenotypic variation for litter size in this cross, an increase of 36% over the direct effects of the QTL. This indicates the importance of epistasis as a component of the genetic architecture of litter size and fitness in our intercross population.

Maternal MTHFR interacts with the offspring's BCL3 genotypes, but not with TGFA, in increasing risk to nonsyndromic cleft lip with or without cleft palate

The 677 C → T polymorphism in the 5-10 methylenetetrahydrofolate reductase (MTHFR) gene has been associated with nonsyndromic cleft lip with or without cleft palate (CL/P) in some populations, but not others. Previous studies (ie, case–control and transmission disequilibrium tests (TDT)) in Brazilian families with CL/P have been unable to replicate this putative association. However, our group observed a lower proportion of CT heterozygotes among the mothers of CL/P probands, suggesting that the maternal genotype for this polymorphism might influence predisposition to CL/P. In order to further examine this issue, we performed a case–control study of the 677 C → T/MTHFR polymorphism in families with CL/P ascertained in two regions of Brazil: 172 from São Paulo (SP) and 252 from Ceará (CE). The control samples included 243 individuals from SP and 401 from CE. TDT was carried out in 102 patients with CL/P and their parents. No evidence of an association was observed between the 677 C → T/MTHFR polymorphism and CL/P using the case–control design, while borderline significance was obtained with the TDT (P=0.055). We have also looked for an interaction between maternal MTHFR genotypes and the propositi offsprings genotypes at two candidate susceptibility loci for CL/P, TGFA and BCL3. Interestingly, we observed an interaction between the maternal MTHFR and offsprings BCL3 genotypes (OR: 2.3; 95% CI: 1.1–4.8; P=0.03) but not with the offsprings TGFA genotypes. Therefore, our results reinforce the idea that the maternal MTHFR genotype plays a significant role in susceptibility to CL/P, but its teratogenic effect depends on the genotype of the offspring.

Analysis of the serotonin transporter polymorphism (5-HTTLPR) in Brazilian patients affected by dysthymia, major depression and bipolar disorder

Analysis of the serotonin transporter polymorphism (5-HTTLPR) in Brazilian patients affected by dysthymia, major depression and bipolar disorder

Species diversity and geographic distribution of Gymnotus (Pisces: Gymnotiformes) by nuclear (GGAC)n microsatellite analysis

No presente estudo foram analisados os padroes de amplificacao de fragmentos de DNA nuclear flanqueados por microssatelites (GGAC)n obtidos a partir de 198 exemplares do genero Gymnotus (Pisces: Gymnotiformes) amostrados em 8 bacias hidrograficas do sudeste brasileiro. As especies analisadas foram Gymnotus carapo, G. pantherinus, G. inaequilabiatus e G. sylvius. Os padroes de amplificacao foram obtidos atraves da tecnica de SPAR (single primer amplification reaction) e refletem, indiretamente, a distribuicao de sequencias repetitivas simples no genoma nuclear dos especimens. Foram encontrados padroes de amplificacao especie-especificos, os quais foram utilizados como potentes ferramentas na analise da distribuicao geografica e diversidade de especies de Gymnotus. Padroes monomorficos foram observados em G. carapo, G. pantherinus e G. inaequilabiatus. Tres padroes polimorficos foram verificados em G. sylvius. Os resultados obtidos atraves da tecnica SPAR indicam que esta e uma abordagem promissora como ferramenta molecular em programas de conservacao de comunidades de peixes de agua doce neotropicais.

Codominant inheritance of polymorphic color variants of Gracilaria domingensis (Gracilariales, Rhodophyta)

Agar is the most valuable phycocoloid in the world market. Currently, about half of its production is obtained from the red alga Gracilaria (Gracilariales, Rhodophyta). Consequently, this genus has been the subject of many studies worldwide. A common green color variant of G. domingensis (Kutzing) Sonder ex Dickie was found in a natural population on the northeastern coast of Brazil. Crosses were performed to determine the mode of color inheritance. The results can be interpreted as the expression of a pair of codominant alleles, where the green and red phenotypes are homozygous, and the heterozygotes present a brownish color. Heterozygous tetrasporophytes, at least until they are 4-5 cm long, exhibit a parental influence on the female gametophyte, since the reddish-brown or greenish-brown color is dependent on the female parent color (red or green). Mixed reproductive phases, as well as specimens with patches of different colors bearing spermatangia or cystocarps, were observed in laboratory cultures. Gametophytes that resulted from in situ germination of meiospores were also observed, and could be easily detected when red or green gametophytes were grown on brown tetrasporophytes.

Diversification of Ramphastinae (Aves, Ramphastidae) prior to the Cretaceous/Tertiary boundary as shown by molecular clock of mtDNA sequences

Partial cytochrome b and 12S rDNA mitochondrial DNA sequences of eight representatives of the Ramphastidae family were analyzed. We applied the linearized tree method to identify sequences evolving at similar rates and estimated the divergence times among some of the taxa analyzed. After excluding Ramphastos tucanus and Capito dayi from our data set, the remaining taxa presented a constant rate of DNA substitution, and branch lengths could be re-estimated with a clock constraint using the maximum likelihood method. Branch lengths were calibrated assuming that Galliformes and Piciformes split around 100 million years ago (mya). Our results indicate that Ramphastinae, and probably Capitoninae, diverged from other Piciformes in the Late Cretaceous (~82 mya), suggesting that Piciformes is another avian order that survived the mass extinction event occurred 65 mya at the Cretaceous/Tertiary (K/T) boundary. The divergence times estimated within the Ramphastinae genera cover the period from the Middle Eocene (around 47 mya) through the Late Miocene (9.5 mya). Our estimate of divergence time is coincidental with the split of the African and the South American continents and other intense geologic activities and modifications of the areas which correspond to the current Neotropics. These events might have influenced the diversification of Ramphastinae in South America.

Group I introns and associated homing endonuclease genes reveals a clinal structure for Porphyra spiralis var. amplifolia (Bangiales, Rhodophyta) along the Eastern coast of South America

BackgroundGroup I introns are found in the nuclear small subunit ribosomal RNA gene (SSU rDNA) of some species of the genus Porphyra (Bangiales, Rhodophyta). Size polymorphisms in group I introns has been interpreted as the result of the degeneration of homing endonuclease genes (HEG) inserted in peripheral loops of intron paired elements. In this study, intron size polymorphisms were characterized for different Porphyra spiralis var. amplifolia (PSA) populations on the Southern Brazilian coast, and were used to infer genetic relationships and genetic structure of these PSA populations, in addition to cox2-3 and rbcL-S regions. Introns of different sizes were tested qualitatively for in vitro self-splicing.ResultsFive intron size polymorphisms within 17 haplotypes were obtained from 80 individuals representing eight localities along the distribution of PSA in the Eastern coast of South America. In order to infer genetic structure and genetic relationships of PSA, these polymorphisms and haplotypes were used as markers for pairwise Fst analyses, Mantels test and median joining network. The five cox2-3 haplotypes and the unique rbcL-S haplotype were used as markers for summary statistics, neutrality tests Tajimas D and Fus Fs and for median joining network analyses. An event of demographic expansion from a population with low effective number, followed by a pattern of isolation by distance was obtained for PSA populations with the three analyses. In vitro experiments have shown that introns of different lengths were able to self-splice from pre-RNA transcripts.ConclusionThe findings indicated that degenerated HEGs are reminiscent of the presence of a full-length and functional HEG, once fixed for PSA populations. The cline of HEG degeneration determined the pattern of isolation by distance. Analyses with the other markers indicated an event of demographic expansion from a population with low effective number. The different degrees of degeneration of the HEG do not refrain intron self-splicing. To our knowledge, this was the first study to address intraspecific evolutionary history of a nuclear group I intron; to use nuclear, mitochondrial and chloroplast DNA for population level analyses of Porphyra; and intron size polymorphism as a marker for population genetics.

The PTPN22 1858T allele but not variants in the proximal promoter region of IL-21 gene is associated with the susceptibility to type 1 diabetes and the presence of autoantibodies in a Brazilian cohort

Interleukin (IL)‐21 and protein tyrosine phosphatase non‐receptor 22 (PTPN22) regulate lymphocyte function and have been implicated in the pathogenesis of autoimmune diabetes. We sequenced the proximal promoter of the IL‐21 gene for the first time and analysed the PTPN22 1858T polymorphism in type 1A diabetes (T1AD) patients and healthy controls (HC). We correlated the frequencies of islet and extra‐pancreatic autoantibodies with genotypes from both loci. The case series comprised 612 T1AD patients and 792 HC. Genotyping of PTPN22 C1858T was performed on 434 T1AD patients and 689 HC. The −448 to +83 base pairs (bp) region of the IL‐21 gene was sequenced in 309 Brazilian T1AD and 189 HC subjects. We also evaluated human leucocyte antigen (HLA) DR3/DR4 alleles. The frequencies of glutamic acid decarboxylase (GAD65), tyrosine phosphatase‐like protein (IA)‐2, anti‐nuclear antibody (ANA), thyroid peroxidase (TPO), thyroglobulin (TG), thyrotrophin receptor autoantibody (TRAb), anti‐smooth muscle (ASM) and 21‐hydroxylase (21‐OH) autoantibodies were higher in T1AD patients than in HC. The PTPN22 1858T allele was associated with an increased risk for developing T1AD [odds ratio (OR) = 1·94; P < 0·001], particularly in patients of European ancestry, and with a higher frequency of GAD65 and TG autoantibodies. HLA‐DR3/DR4 alleles predominated in T1AD patients. A heterozygous allelic IL‐21 gene variant (g.‐241 T > A) was found in only one patient. In conclusion, only PTPN22 C1858T polymorphism and HLA‐DR3 and/or DR4 alleles, but not allelic variants in the 5′‐proximal region of the IL‐21 gene were associated with T1AD risk. Patients with T1AD had increased frequencies of anti‐islet‐cell, anti‐thyroid, anti‐nuclear, anti‐smooth muscle and anti‐21‐OH autoantibodies. The C1858T PTPN22 polymorphism was also associated with a higher frequency of GAD65 and TG autoantibodies.