Research Article

Table of Contents | Genet. Mol. Res. 2022 (3)

Displaying 1 - 6 of 6
Research Article

Spontaneous abortions, which occur in 15-20% of all pregnancies, constitute the most common human genetic disease, since most miscarriages are caused by chromosomal anomalies. In our laboratory, we devised a protocol for sequential use of quantitative fluorescent PCR (QF-PCR), homologous gene quantitative PCR (HGQ-PCR) and SNP-arrays that allows examination of numerical and structural analyses of all chromosomes in spontaneous abortions at low cost. We describe our results with 1,295 samples of fetal tissues collected consecutively after pregnancy losses. Positive signals with QF-PCR and HGQ-PCR were always confirmed using microsatellite amplification of the specific chromosome involved.  Among the exams, 64.6% were abnormal. The most common anomalies were trisomies (69.5%), triploidy (13.5%) and monosomy X (9.1%). The most frequent trisomies involved the following chromosomes: 16 (23.5%), 22 (16.0%), 21 (14.1%) and 15 (10.1%). SNP array analysis permitted the diagnoses of all trisomies. Additionally, deletions and/or duplications and chromosomal mosaicism were detected by SNP-array in 23 cases. In conclusion, our sequential analysis of fetal tissues is a new, highly useful, rapid, and cost-effective approach for the diagnosis of chromosomal alterations in spontaneous abortions.

Genet. Mol. Res. 21(3): GMR19082
DOI: 10.4238/gmr19082
Research Article

The Brazilian Testudinidae family is widespread across South America. It includes Chelonoidis denticulatus, the largest tortoise in South America and Chelonoidis carbonarius, found mostly in the north and northwestern part of the continent. Using hemoglobin to identify species is cheaper than other methods such as DNA sequencing and can offer useful information, since the hemoglobin molecule is a well-preserved protein chain during the evolution of species. Thus, in order to establish a hemoglobin profile for the Brazilian Testudinidae C. denticulatus, C. carbonarius and morphotype 1, hemoglobin electrophoresis was performed at acid pH in phosphate agar and at alkaline pH in cellulose acetate, in order to visualize the specific fractions of each species. High performance liquid chromatography was used for the quantification of fractions. For an in-depth analysis and better detailing of the hemoglobin profile of the species, polypeptide chain electrophoresis was performed at acid and alkaline pH. We observed differences in the hemoglobin profiles of C. denticulatus in relation to C. carbonarius and morphotype 1, which suggests that this methodology, not common in taxonomic studies, can help determine relationships between species, since hemoglobins are proteins with well-preserved genes. We found differences in hemoglobin mobility between C. denticulatus, C. carbonarius and morphotype 1 in electrophoresis at alkaline pH, however, the behavior of globin chains was similar between the three groups. High performance liquid chromatography showed different retention times in the globin fractions of C. denticulatus and C. carbonarius, but not between C. carbonarius and morphotype 1, indicating that, possibly, the divergence time between C. carbonarius and morphotype 1 is more recent than the divergence between C. denticulatus and C. carbonarius, due to the highly conserved character of this functional protein. Thus, considering the high degree of conservation of hemoglobins in vertebrates, and the differences observed in electrophoresis at alkaline pH and HPLC, we infer that C. carbonarius and morphotype 1 present a common branch.

Genet. Mol. Res. 21(3): GMR18977
DOI: 10.4238/gmr18977
Research Article

Research on genes affecting phenotypic variation in milk production and composition from indicine (Bos indicus) cattle is imperative, since these breeds are important tropical genetic resources, and there have been few studies investigating the genetic basis of these traits. We identified polymorphisms in k-casein (CSN3), b-lactoglobulin (LGB), thyroglobulin (TG) and prolactin (PRL) and examined their effect on milk and composition traits in the Guzerá breed. DNA samples of 260 Guzerá  cattle selected for dual purpose use were genotyped. Allele frequencies observed for the A allele were 0.83, 0.18 and 0.25 respectively for CSN3, LGB and PRL genes, while for the TG gene T allele had an allele frequency of 0.09. For all polymorphisms evaluated, observed genotypic frequencies were in agreement with those expected according to the Hardy-Weinberg Equilibrium hypothesis. A polymorphism association study evaluated breeding values (BV) for 305-day milk (BV-M), fat (BV-F), and protein (BV-P) production, employing the allele substitution model using a sample of 139 cows belonged to 27 full and half-sib families of a MOET (multiple ovulation and embryo transfer) selection nucleus. Association was found between the LGB polymorphism and BV-M, BV-F and BV-P. Animals with LGB AA genotype have, on average, higher BV when compared to animals with LGB AB and BB genotypes (277.85 kg for BV-M, 12.09 kg for BV-F and 9.33 kg for BV-P).  These findings contribute to a better understanding on the influence of these polymorphisms on milk production traits in Guzerá cattle.

Genet. Mol. Res. 21(3): GMR19046
DOI: 10.4238/gmr19046
Research Article

We developed a new and simple feeding device for Drosophila melanogaster. In addition, we tested three negative geotaxis methods (measuring the percentage of the flies able to climb 8 cm in 8 s, measuring the distance climbed in 3 s, and measuring the distance climbed in 8 s). The flies were exposed to chlorpyrifos (CPF) using the new feeding device. Our results demonstrated that the three methods for measuring negative geotaxis could be used interchangeably with respect to the needs and conditions of the experiments; however, we recommend the 8 s method with PAST software because the other two methods were carried out using manual measurements. The use of this free software makes the process more accurate with no additional cost. We found that CPF caused impairment in locomotor activity, reduction in AChE activity, and disturbance of the dopaminergic pathways in D. melanogaster, suggesting that CPF toxicity is not confined to the cholinergic system. This study provides a new system to study neurodegenerative damage using a user-friendly and no-cost software for measuring climbing activity in D. melanogaster.

Genet. Mol. Res. 21(3): GMR19056
DOI: 10.4238/gmr19056
Research Article

The dehydration responsive element (DRE)-binding proteins (DREB) play a role in the signaling network that activates many abiotic stress-responsive genes. We isolated and molecularly characterized the DREB gene from ancestral diploid wheat species growing in Azerbaijan and native to this region. This territory is included in a region that is considered the center of origin of cultivated wheat. One-week-old seedlings of Triticum urartu (Au), Aegilops speltoides (B) and Ae. tauschii (D) were used for genomic DNA extraction. Gene-specific primer pairs were applied forisolation of the DREB gene. The amplification products were purified using a gel extraction kit and sequenced. Data analysis was performed using FGENESH, BLAST, INTERPROSCAN, SMART, MAFFT, ExPASy, ProteinPredict, and PSIPRED tools. Ensembl Plants and NCBI were used as integrative resources. Numerous SNPs and nine microindels were detected in the partial target sequence of the DREB gene in Ae. speltoides. Nonsynonymous SNPs were determined in T. urartu (1 transition and 5 transversions), and Ae. tauschii (2 transitions and 2 transversions). Analysis of amino acid sequences encoded by the putative DREB genes revealed a conserved AP2/ERF domain, with two conserved functional amino acids (14th valine and 19th glutamic acid) that play crucial roles in the recognition of the DNA-binding sequence and two tryptophan rings that determine the geometry of the GCC-box binding domain. Nuclear localization signal and conserved Ser/Thr-rich region were observed in the corresponding amino acid sequences. One α-helix and three β-sheets were detected in the secondary structure of the AP2 domain. The isolated sequences of DREB gene from T. urartu and Ae. tauschii were confirmed and registered in NCBI with Accession Numbers MZ935739 and MZ935740. Identification of the DREB gene in wheat progenitors and its characterization is important for evaluating their genomic material for possible use to enhance the diversity of wheat cultivated under stress conditions.

Genet. Mol. Res. 21(3): GMR19035
DOI: 10.4238/gmr19035
Research Article

Quantitative PCR puts great demands on DNA quality and relies on a comparison of fragment amplification between two chromosomes using different primers. The use of a single primer pair capable of reliable relative comparative amplification would be a great advantage.  Using this approach, we developed a rapid, high-throughput, semi-automated and cost-efficient multiplexed method for molecular determination of chromosomal sex called MQS-PCR – multiplex quantitative sexing PCR. DNA sequences located on different chromosomes and differing in length can be amplified and fluorescently labelled with a common fluorescent primer and conveniently separated and detected using capillary electrophoresis. In this method, the intensity of amplification of each of the fragments is compared to determine DNA dosage. MQS-PCR achieves 100% analytical sensitivity and specificity in detecting normal and abnormal sex chromosomal complements (45,X, 46,X,i(X)(q10), 46,X,i(X)(p10), 46,X,X(p-), 46,X,X(q-), 47,XXX, 47,XXY and 47,XYY). It is a reliable, low cost, and rapid detection method for the determination of chromosomal sex and sex chromosomal abnormalities in human samples.

Genet. Mol. Res. 21(3): GMR19067
DOI: 10.4238/gmr19067