SSCP-polymorphism in intron 12 of the CFTR gene recognized by BclI

Source/Description: SSCP analysis of intron 12 of the CFTR gene from PCR products showed an extra band in several DNA samples. Sequencing of the additional fragment extra band revealed a T- A change in the position 1898 + 152 of CFTR (Fig. 1). The change is a polymorphism which can be identified by SSCP or by BclI digestion...

Adaptive colour polymorphism of Acrida ungarica H. (Orthoptera: Acrididae) in a spatially heterogeneous environment

Intra-specific colour polymorphism provides a cryptic camouflage from predators in heterogeneous habitats. The orthoptera species, Acrida ungarica (Herbst, 1786) possess two well-distinguished colour morphs: brown and green and displays several disruptive colouration patterns within each morph to improve the crypsis. This study focused on how the features of the background environment relate to the proportion of the two morphs and to the intensity of disruptive colouration patterns in A. ungarica. As the two sexes are very distinct with respect to mass and length, we also distinctively tested the relationship for each sex. In accordance with the background matching hypothesis, we found that, for both sexes, the brown morph was in higher proportion at sites with a brown-dominant environment, and green morphs were in higher proportion in green-dominant environments. Globally, individuals in drier sites and in the drier year also had more intense disruptive colouration patterns, and brown morphs and females were also more striped. Colour patterns differed largely between populations and were significantly correlated with relevant environmental features. Even if A. ungarica is a polymorphic specialist, disruptive colouration still appears to provide strong benefits, particularly in some habitats. Moreover, because females are larger, they are less able to flee, which might explain the difference between sexes

A polymorphic microsatellite in the tumor necrosis factor alpha promoter identifies an allele unique to the NZW mouse strain.

We have amplified a (CA)n:(GT)n microsatellite from the TNF promoters of a panel of mouse strains using the polymerase chain reaction. The length of the microsatellites was polymorphic, with eight alleles observed among 15 inbred strains bearing seven distinct H-2 haplotypes, and four outbred strains. In B10 congenic strains, the TNF allele detected by microsatellite polymorphism segregated with the MHC, and in recombinant haplotypes (NOD, NZW), it segregated with H-2D. The TNF allele found in the NZW strain (H-2z) was distinct from those of all other haplotypes, consistent with the hypothesis that this strain may carry a genetic defect in TNF production.

Genetic variability of Brazilian phytoplasma and spiroplasma isolated from maize plants

The objective of this work was to characterize the genetic variability of phytoplasma and Spiroplasma kunkelii isolated from maize plants showing symptoms of stunt collected from different Brazilian geographic regions. A DNA fragment of 500 base pairs (bp) was amplified from the spiralin gene in S. kunkelii and one fragment of 1,200 bp was generated from 16S rDNA gene in phytoplasma. The partial sequences of the spiralin gene showed similarity of 98% among the isolates of S. kunkelii analyzed. These sequences were compared with the sequence of the spiralin gene from other Spiroplasma species deposited in the GenBank, resulting in a similarity varying from 76.9% to 88.1%. The 16S rDNA sequence from the phytoplasma were completely similar within the Brazilian isolates and showed up to 98% of the similarity with sequences already found from other phytoplasmas. A very narrow genetic variability was detected by these gene fragments within phytoplasma and Spiroplasma analyzed. However, other genomic regions with higher polymorphic levels shall be identified in order to better evaluate the genetic diversity within these microorganisms population.

Length variation in HIV-1 gp120 as the product of DNA misalignment mechanism

Background: To determine whether misalignment structures such as duplications, repeats, and palindromes are associated to insertions/deletions (indels) in gp120, indicating that indels are indeed frameshift mutations generated by DNA misalignment mechanism. Methods: Cloning and sequencing of a fragment of HIV-1 gp120 spanning C2-C4 derived from plasma RNA in 12 patients with early chronic disease and naïve to antiretroviral therapy. Results: Indels in V4 involved always insertion and deletion of duplicated nucleotide segments, and AAT repeats, and were associated to the presence of palindromic sequences. No duplications were detected in V3 and C3. Palindromic sequences occurred with similar frequencies in V3, C3 and V4; the frequency of palindromes in individual genes was found to be significantly higher in structural (gp120, p ≤ 3.00E-7) and significantly lower in regulatory (Tat, p ≤ 9.00E-7) genes, as compared to the average frequency calculated over the full genome. Discussion: Indels in V4 are associated to misalignment structures (i.e. duplications repeat and palindromes) indicating DNA misalignment as the mechanism underlying length variation in V4. The finding that indels in V4 are caused by DNA misalignment has some very important implications: 1) indels in V4 are likely to occur in proviral DNA (and not in RNA), after integration of HIV into the host genome; 2) they are likely to occur as progressive modifications of the early founder virus during chronic infection, as more and more cells get infected; 3) frameshift mutations involving any number of base pairs are likely to occur evenly across gp120; however, only those mutants carrying a functional gp120 (indels as multiples of three base pairs) will be able to perpetuate the virus cycle and to keep spreading through the population.

Mutant huntingtin impairs the post-Golgi trafficking of brain-derived neurotrophic factor but not its Val66Met polymorphism.

Brain-derived neurotrophic factor (BDNF) polymorphism is associated with the pathophysiology of several neurodegenerative disorders, including Huntington"s disease. In view ofthese data andthe involvement of huntingtin in intracellular trafficking, we examined the intracellular transport and release of Val66Val BDNF (Val-BDNF) and Val66Met BDNF (Met-BDNF) in transfected striatal knock-in cells expressing wild-type or mutant full-length huntingtin. Colocalization studies with specific markers for endoplasmic reticulum showed no differences between the Val-BDNF and Met-BDNF and were not modified by mutant huntingtin. However, post-Golgi trafficking was altered by mutant huntingtin dependent on the BDNF form. Thus, fluorescence recovery after photobleaching (FRAP) and inverse FRAP analysis showed retention of Met-BDNF inthe Golgi apparatus with respectto Val-BDNF in wild-type cells. Strikingly, mutant huntingtin diminished post-Golgi trafficking of Val-BDNF, whereas Met-BDNF was not modified. Accordingly, a reduction in the number of transport vesicles was only observed in mutant huntingtin cells transfected with Val-BDNF but not Met-BDNF. Moreover, mutant huntingtin severely affectedthe KCl-evoked release of Val-BDNF, although it had little effect on Met-BDNF regulated release. The constitutive release of Val-BDNF or Met-BDNF in mutant cells was only slightly reduced. Interestingly, mutant huntingtin only perturbed post-Golgi trafficking of proteins that follow the regulated secretory pathway (epidermal growth factor receptor or atrial natriuretic factor), whereas it did not change those that follow the constitutive pathway (p75 NTR ). We conclude that mutant huntingtin differently affects intracellular transport and release of Val-BDNF and Met-BDNF. In addition, our findings reveal a new role for huntingtin in the regulation of the post-Golgi trafficking of the regulated secretory pathway.

Reversal of a full-length mutant huntingtin neuronal cell phenotypeby chemical inhibitors of polyglutamine-mediated aggregation

Background: Huntington's disease (HD) is an inherited neurodegenerative disorder triggered by an expanded polyglutamine tract in huntingtin that is thought to confer a new conformational property on this large protein. The propensity of small amino-terminal fragments with mutant, but not wild-type, glutamine tracts to self-aggregate is consistent with an altered conformation but such fragments occur relatively late in the disease process in human patients and mouse models expressing full-length mutant protein. This suggests that the altered conformational property may act within the full-length mutant huntingtin to initially trigger pathogenesis. Indeed, genotypephenotype studies in HD have defined genetic criteria for the disease initiating mechanism, and these are all fulfilled by phenotypes associated with expression of full-length mutant huntingtin, but not amino-terminal fragment, in mouse models. As the in vitro aggregation of amino-terminal mutant huntingtin fragment offers a ready assay to identify small compounds that interfere with the conformation of the polyglutamine tract, we have identified a number of aggregation inhibitors, and tested whether these are also capable of reversing a phenotype caused by endogenous expressionof mutant huntingtin in a striatal cell line from the HdhQ111/Q111 knock-in mouse. Results: We screened the NINDS Custom Collection of 1,040 FDA approved drugs and bioactive compounds for their ability to prevent in vitro aggregation of Q58-htn 1¿171 amino terminal fragment. Ten compounds were identified that inhibited aggregation with IC50 < 15 ¿M, including gossypol, gambogic acid, juglone, celastrol, sanguinarine and anthralin. Of these, both juglone and celastrol were effective in reversing the abnormal cellular localization of full-length mutant huntingtin observed in mutant HdhQ111/Q111 striatal cells. Conclusions: At least some compounds identified as aggregation inhibitors also prevent a neuronal cellular phenotype caused by full-length mutant huntingtin, suggesting that in vitro fragment aggregation can act as a proxy for monitoring the disease-producing conformational property in HD. Thus, identification and testing of compounds that alter in vitro aggregation is a viable approach for defining potential therapeutic compounds that may act on the deleterious conformational property of full-length mutant huntingtin.

Neuropeptide Y at the cellular level – Studies on PreproNPY L7P Polymorphism and the Mitochondrial Form of NPY

Neuropeptide Y (NPY) is a widely expressed neurotransmitter in the central and peripheral nervous systems. Thymidine 1128 to cytocine substitution in the signal sequence of the preproNPY results in a single amino acid change where leucine is changed to proline. This L7P change leads to a conformational change of the signal sequence which can have an effect on the intracellular processing of NPY. The L7P polymorphism was originally associated with higher total and LDL cholesterol levels in obese subjects. It has also been associated with several other physiological and pathophysiological responses such as atherosclerosis and T2 diabetes. However, the changes on the cellular level due to the preproNPY signal sequence L7P polymorphism were not known. The aims of the current thesis were to study the effects of the [p.L7]+[p.L7] and the [p.L7]+[p.P7] genotypes in primary cultured and genotyped human umbilical vein endothelial cells (HUVEC), in neuroblastoma (SK-N-BE(2)) cells and in fibroblast (CHO-K1) cells. Also, the putative effects of the L7P polymorphism on proliferation, apoptosis and LDL and nitric oxide metabolism were investigated. In the course of the studies a fragment of NPY targeted to mitochondria was found. With the putative mitochondrial NPY fragment the aim was to study the translational preferences and the mobility of the protein. The intracellular distribution of NPY between the [p.L7]+[p.L7] and the [p.L7]+[p.P7] genotypes was found to be different. NPY immunoreactivity was prominent in the [p.L7]+[p.P7] cells while the proNPY immunoreactivity was prominent in the [p.L7]+[p.L7] genotype cells. In the proliferation experiments there was a difference in the [p.L7]+[p.L7] genotype cells between early and late passage (aged) cells; the proliferation was raised in the aged cells. NPY increased the growth of the cells with the [p.L7]+[p.P7] genotype. Apoptosis did not seem to differ between the genotypes, but in the aged cells with the [p.L7]+[p.L7] genotype, LDL uptake was found to be elevated. Furthermore, the genotype seemed to have a strong effect on the nitric oxide metabolism. The results indicated that the mobility of NPY protein inside the cells was increased within the P7 containing constructs. The existence of the mitochondria targeted NPY fragment was verified, and translational preferences were proved to be due to the origin of the cells. Cell of neuronal origin preferred the translation of mature NPY (NPY1-36) when compared to the non neuronal cells that translated both, NPY and the mitochondrial fragment of NPY. The mobility of the mitochondrial fragment was found to be minimal. The functionality of the mitochondrial NPY fragment remains to be investigated. L7P polymorphism in the preproNPY causes a series of intracellular changes. These changes may contribute to the state of cellular senescence, vascular tone and lead to endothelial dysfunction and even to increased susceptibility to diseases, like atherosclerosis and T2 diabetes.

Detection and coat protein gene characterization of an isolate of Grapevine virus B from corky bark-affected grapevines in Southern Brazil

An isolate of Grapevine virus B (GVB), obtained by indexing Vitis labrusca and V. vinifera grapevines on the indicator LN33, was transmitted mechanically to several Nicotiana species. The virus was partially purified from N. cavicola and the coat protein estimated at 23 kDa by SDS-PAGE. In negatively stained leaf extracts of experimentally inoculated N. cavicola and N. occidentalis, flexuous particles with cross banding were observed, predominantly measuring 750-770 x 12 nm, with a modal length of 760 nm. Decoration indicated a clear, positive reaction against AS-GVB. In DAS-ELISA, GVB was detected in N. cavicola and grapevine extracts, and Western blots showed homologous and cross reaction of GVB and GVA antisera with GVB coat protein. Using specific primers for GVB, a fragment of 594 bp, comprising the coat protein gene coding for 197 amino acids, was amplified by RT-PCR with viral RNA extracted from GVB-infected N. occidentalis. The nucleotide and the deduced amino acid sequences of the coat protein gene showed high identities with Italian and Japanese isolates of GVB.

Detection and characterization of fibropapilloma associated herpesvirus of marine turtles in Rio Grande do Sul, Brazil

Fibropapillomatosis (FP) is a benign tumoral disease that affects sea turtles, hampering movement, sight and feeding, ultimately leading to death. In Brazil, the disease was described for the first time in 1986. Research suggests the involvement of a herpesvirus in association with environmental and genetic factors as causal agents of FP. The objective of the present study was to detect and characterize this herpesvirus in sea turtles living in the coast of state Rio Grande do Sul (RS), Brazil. From October 2008 to July 2010, 14 turtles were observed between the beaches of Torres and Tavares, of which 11 were green turtles (Chelonia mydas) and 3 were loggerhead turtles (Caretta caretta). All turtles were young and mean curved carapace length was 37.71±7.82cm, and varied from 31 to 55cm. Only one green turtle presented a 1cm, papillary, pigmented fibropapilloma. Skin and fibropapilloma samples were analyzed by conventional and real time PCR assays to detect and quantify herpesvirus. All skin samples were negative, though the fibropapilloma specimen was positive in both tests. Viral load was 9,917.04 copies of viral genome per milligram of tissue. The DNA fragment amplified from the fibropapilloma sample was sequenced and allocated in the Atlantic phylogeographic group. This study reports the first molecular characterization of herpesvirus associated with fibropapilloma in turtles from the coast of RS.

Association of apolipoprotein E polymorphism in late-onset Alzheimer's disease and vascular dementia in Brazilians

The genetic basis for dementias is complex. A common polymorphism in the apolipoprotein E (APOE) gene is considered to be the major risk factor in families with sporadic and late-onset Alzheimer's disease as well as in the general population. The distribution of alleles and genotypes of the APOE gene in late-onset Alzheimer's disease (N = 68), other late-life dementias (N = 39), and in cognitively normal controls (N = 58) was determined, as also was the risk for Alzheimer's disease associated with the epsilon4 allele. Peripheral blood samples were obtained from a total of 165 individuals living in Brazil aged 65-82 years. Genomic DNA was amplified by the polymerase chain reaction and the products were digested with HhaI restriction enzyme. APOE epsilon2 frequency was considerably lower in the Alzheimer's disease group (1%), and the epsilon3 allele and epsilon3/epsilon3 genotype frequencies were higher in the controls (84 and 72%, respectively) as were the epsilon4 allele and epsilon3/epsilon4 genotype frequencies in Alzheimer's disease (25 and 41%, respectively). The higher frequency of the epsilon4 allele in Alzheimer's disease confirmed its role as a risk factor, while epsilon2 provided a weak protection against development of the disease. However, in view of the unexpectedly low frequency of the epsilon4 allele, additional analyses in a more varied Brazilian sample are needed to clarify the real contribution of apolipoprotein E to the development of Alzheimer's disease in this population.

Mutations in SRY and WT1 genes required for gonadal development are not responsible for XY partial gonadal dysgenesis

The WT1 transcription factor regulates SRY expression during the initial steps of the sex determination process in humans, activating a gene cascade leading to testis differentiation. In addition to causing Wilms' tumor, mutations in WT1 are often responsible for urogenital defects in men, while SRY mutations are mainly related to 46,XY pure gonadal dysgenesis. In order to evaluate their role in abnormal testicular organogenesis, we screened for SRY and WT1 gene mutations in 10 children with XY partial gonadal dysgenesis, 2 of whom with a history of Wilms' tumor. The open reading frame and 360 bp of the 5' flanking sequence of the SRY gene, and the ten exons and intron boundaries of the WT1 gene were amplified by PCR of genomic DNA. Single-strand conformation polymorphism was initially used for WT1 mutation screening. Since shifts in fragment migration were only observed for intron/exon 4, the ten WT1 exons from all patients were sequenced manually. No mutations were detected in the SRY 5' untranslated region or within SRY open-reading frame sequences. WT1 sequencing revealed one missense mutation (D396N) in the ninth exon of a patient who also had Wilms' tumor. In addition, two silent point mutations were found in the first exon including one described here for the first time. Some non-coding sequence variations were detected, representing one new (IVS4+85A>G) and two already described (-7ATG T>G, IVS9-49 T>C) single nucleotide polymorphisms. Therefore, mutations in two major genes required for gonadal development, SRY and WT1, are not responsible for XY partial gonadal dysgenesis.

Frequency of Werner helicase 1367 polymorphism and age-related morbidity in an elderly Brazilian population

Werner syndrome (WS) is a premature aging disease caused by a mutation in the WRN gene. The gene was identified in 1996 and its product acts as a DNA helicase and exonuclease. Some specific WRN polymorphic variants were associated with increased risk for cardiovascular diseases. The identification of genetic polymorphisms as risk factors for complex diseases affecting older people can improve their prevention, diagnosis and prognosis. We investigated WRN codon 1367 polymorphism in 383 residents in a district of the city of São Paulo, who were enrolled in an Elderly Brazilian Longitudinal Study. Their mean age was 79.70 ± 5.32 years, ranging from 67 to 97. This population was composed of 262 females (68.4%) and 121 males (31.6%) of European (89.2%), Japanese (3.3%), Middle Eastern (1.81%), and mixed and/or other origins (5.7%). There are no studies concerning this polymorphism in Brazilian population. These subjects were evaluated clinically every two years. The major health problems and morbidities affecting this cohort were cardiovascular diseases (21.7%), hypertension (83.7%), diabetes (63.3%), obesity (41.23%), dementia (8.0%), depression (20.0%), and neoplasia (10.8%). Their prevalence is similar to some urban elderly Brazilian samples. DNA was isolated from blood cells, amplified by PCR and digested with PmaCI. Allele frequencies were 0.788 for the cysteine and 0.211 for the arginine. Genotype distributions were within that expected for the Hardy-Weinberg equilibrium. Female gender was associated with hypertension and obesity. Logistic regression analysis did not detect significant association between the polymorphism and morbidity. These findings confirm those from Europeans and differ from Japanese population.

TP53 codon 72 polymorphism as a risk factor for cardiovascular disease in a Brazilian population

TP53, a tumor suppressor gene, has a critical role in cell cycle, apoptosis and cell senescence and participates in many crucial physiological and pathological processes. Identification of TP53 polymorphism in older people and age-related diseases may provide an understanding of its physiology and pathophysiological role as well as risk factors for complex diseases. TP53 codon 72 (TP53:72) polymorphism was investigated in 383 individuals aged 66 to 97 years in a cohort from a Brazilian Elderly Longitudinal Study. We investigated allele frequency, genotype distribution and allele association with morbidities such as cardiovascular disease, type II diabetes, obesity, neoplasia, low cognitive level (dementia), and depression. We also determined the association of this polymorphism with serum lipid fractions and urea, creatinine, albumin, fasting glucose, and glycated hemoglobin levels. DNA was isolated from blood cells, amplified by PCR using sense 5'-TTGCCGTCCCAAGCAATGGATGA-3' and antisense 5'-TCTGGGAAGGGACAGAAGATGAC-3' primers and digested with the BstUI enzyme. This polymorphism is within exon 4 at nucleotide residue 347. Descriptive statistics, logistic regression analysis and Student t-test using the multiple comparison test were used. Allele frequencies, R (Arg) = 0.69 and P (Pro) = 0.31, were similar to other populations. Genotype distributions were within Hardy-Weinberg equilibrium. This polymorphism did not show significant association with any age-related disease or serum variables. However, R allele carriers showed lower HDL levels and a higher frequency of cardiovascular disease than P allele subjects. These findings may help to elucidate the physiopathological role of TP53:72 polymorphism in Brazilian elderly people.

Apolipoprotein CIII polymorphism and triglyceride levels of a Japanese population living in Southern Brazil

Apolipoprotein CIII (apo-CIII) participates in the regulation of triglyceride-rich lipoprotein metabolism. Several polymorphic sites have been detected within and around the apo-CIII gene. Here, we examined the relationship between apo-CIII SstI polymorphism (CC, CG, GG genotypes) and plasma triglyceride (TG) levels in a group of 159 Japanese individuals living in Southern Brazil. The sample was divided into a group of Japanese descendants (N = 51) with high TG (HTG; >200 mg/dL) and a group of Japanese descendants (N = 108) with normal TG (NTG; <200 mg/dL). TG and total cholesterol levels were analyzed by an enzymatic method using the Labtest-Diagnostic kit and high- and low-density lipoproteins by a direct method using the Labtest-Diagnostic kit and DiaSys Diagnostic System International kit, respectively. A 428-bp sequence of apo-CIII gene was amplified using oligonucleotide primers 5' GGT GAC CGA TGG CTT CAG TTC CCT GA 3' and 5' CAG AAG GTG GAT AGA GCG CTG GCC T 3'. The PCR products were digested with a restriction endonuclease SstI. Rare G allele was highly prevalent in our study population (0.416) compared to Caucasians (0.00-0.11). G allele was almost two times more prevalent in the HTG group compared to the NTG group (P < 0.001). The genotype distribution was consistent with the Hardy-Weinberg equilibrium. There was a significant association between rare G allele and HTG in Japanese individuals living in Southern Brazil as indicated by one-way ANOVA, P < 0.05.