No Association of Prolactin Receptor (PRLR) Gene and litter size in Gaddi Goat Breed Reared under Migratory System in Western Himalayan Ranges of Himachal Pradesh

Gaddi is the predominant Indian goat breed also known as “White Himalayan goat”, constituting 60-65% of total goats in the state of Himachal Pradesh. The polymorphism of prolactin receptor (PRLR) gene was found to have relationship with prolificacy in goats. In present study, polymorphism of intron 2 region of PRLR gene was investigated in Gaddi goats (n = 89) using PCR-SSCP and DNA sequencing approach. PCR-SSCP assay of 176 bp amplicon of intron 2 region of PRLR gene revealed polymorphism with three types of genotypes viz., AA, AB and BB with genotypic frequencies as 0.31, 0.55 and 0.14, respectively. The allelic frequency of alleles A and B were 0.59 and 0.41, respectively in all the screened goat population. Genetic diversity analysis revealed the value of Ne, Hobs, Hexp and PIC were 1.96, 0.52, 0.49 and 0.37, respectively. The Ne and Hobs values also indicated that sufficient genetic variation exists at the studied locus. FIS estimate was observed as -0.15 indicating heterozygous excess at studied locus. DNA sequencing of amplified product revealed one nucleotide mutation (T92C) in intron 2 region of PRLR gene. The mean litter size in AA, AB and AB genotypes were 1.27±0.12, 1.41±0.09 and 1.84±0.26, respectively. No significant (P>0.05) associations of PRLR genotypes with litter size were observed. Effect of season and parity were also found to be non-significant (P>0.05) on litter size. Consequently, the study on additional data based on more number of animals in diversified flock should be carried out for future association studies.

Estudio molecular del exón 3 del gen atp7b en pacientes cubanos con Enfermedad de Wilson / Molecular genetic analysis of exon 3 of the ATP7B gene in Cuban patients with Wilson's disease

Introducción: La Enfermedad de Wilson es una enfermedad con patrón de herencia autosómico recesivo. Es causada por las mutaciones en el gen atp7b. El exón 3 del gen atp7b es polimórfico y se informan más de 120 polimorfismos en el gen atp7b. Objetivo: Identificar los cambios conformacionales en el exón 3 del gen atp7b y detectar polimorfismos en pacientes cubanos con diagnóstico clínico presuntivo de la enfermedad de Wilson. Materiales y Métodos: Se realizó un estudio descriptivo, en el Centro Nacional de Genética Médica y en el Instituto Nacional de Gastroenterología, durante el período 2007-2013, que incluyó 105 pacientes con diagnóstico clínico presuntivo de la enfermedad de Wilson. La extracción del ADN fue por la técnica de precipitación salina. Se utilizó la técnica de Reacción en Cadena de la Polimerasa para la amplificación del fragmento de interés, y para detectar los cambios conformacionales y la presencia del polimorfismo p.L456V, se usó la técnica de Polimorfismo Conformacional de Simple Cadena, en el exón 3 del gen atp7b. Resultados: En el exón 3 se detectan los cambios conformacionales denominados b y c que correspondieron al polimorfismo p.L456V en estado heterocigótico y homocigótico respectivamente. La frecuencia alélica del polimorfismo p.L456V es de 41 por ciento. Las manifestaciones más frecuentes en los pacientes que presentaron este polimorfismo son las hepáticas. Conclusiones: Se identificó el polimorfismo p.L456V en 64 pacientes cubanos con diagnóstico clínico de la enfermedad de Wilson, lo cual posibilitará hacer estudios moleculares por métodos indirectos(AU)

Introduction: Wilson's disease is a rare inherited autosomal recessive disorder caused by mutations in the ATP7B gene. The exon 3 of the ATP7B gene is polymorphic, and more than 120 polymorphisms of this type have been reported in the literature. Objective: To identify conformational band shifts in exon 3 and detect polymorphisms of the ATP7B gene in Cuban patients, clinically diagnosed with Wilson's disease. Materials and Methods: A descriptive study including 105 patients with the clinical diagnosis of Wilson's disease was conducted at the National Center for Medical Genetics and the National Institute of Gastroenterology from 2007 to 2013. Salting-out protocol was used for DNA extraction. The Polymerase Chain Reaction was used to amplify the fragment of interest and the Single-Strand Conformational Polymorphism was applied in the region of exon 3 of the ATP7B gene to identify conformational changes and the presence of the polymorphism p.L456V. Results: The conformational change called B and C corresponded to the p.L456V polymorphism in the heterozygous and homozygous states, respectively. The allelic frequency of the p.L456V polymorphism in 105 Cuban patients clinically diagnosed with Wilson's disease was 41 percent. The most common manifestations in patients with this polymorphism were related to the liver. Conclusion: The p.L456V polymorphism was identified in 64 Cuban patients with Wilson disease, which will enable us to conduct molecular studies by indirect methods(AU)

Determination of Repeat Numbers of (CA)n in Mitochondrial D-loop using Polymerase Chain Reaction-single Strand Conformational Polymorphism (PCR-SSCP) / 체질인류학회지

Polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) analysis is a kind of sensitive mutation detection method that has been usually used in field of medical genetics. A single DNA strand with a mutation or nucleotide polymorphism has a different conformation from its wild-type counterpart, and these conformational differences result in different electrophoretic mobility. In previous study of mitochondrial microsatellite instability in 50 uterine leiomyomas, PCR-SSCP showed 4 types of band mobility at (CA)n of the mitochondrial D-loop. In type 1 and 4, positions of the lower single stand of both were same but those of upper strand were different. In sequencing analysis, repeat number of (CA)n in type 1 was 4, 5 in type 2, 6 in type 3, and 4 in type 4, respectively. Without using expensive sequencing analysis, PCR-SSCP method can be used to detect the repeat number of (CA)n in mitochondrial D-loop.

Análisis de los polimorfismos p. K832R y p. T991T en pacientes cubanos con diagnóstico clínico de la enfermedad de Wilson / p. K832R and p. T991T polymorphisms’ analysis in Cuban patients with clinical diagnosis of Wilson's disease

Introducción: La Enfermedad de Wilson se caracteriza por la acumulación de cobre en hígado, cerebro, riñones y cornea. Se transmite con un patrón de herencia autosómico recesivo. La causa molecular que la provoca son las mutaciones en el gen ATP7B. Se han informado en la literatura más de 139 polimorfismos en el gen ATP7B. Objetivo: Identificar los cambios conformacionales en los exones 10 y 13 y detectar los polimorfismos p.K832R y p.T991T en el gen ATP7B en pacientes cubanos con diagnóstico clínico de Enfermedad de Wilson. Material y Métodos: Se realizó un estudio descriptivo, durante el período 2012 al 2013, que incluyó 27 pacientes con diagnóstico clínico de Enfermedad de Wilson. Para la amplificación del fragmento de interés, se utilizó la técnica de Reacción en Cadena de la Polimerasa y para identificar los cambios conformacionales se aplicó la técnica de Polimorfismo Conformacional de Simple Cadena, en el exón 10 y 13 del gen ATP7B. La presencia de los polimorfismos p.K832R y p.T991T fueron identificados por secuenciación. Resultados: Se detectaron tres cambios conformacionales diferentes denominados: (a, b y c) en el exón 10 y (a y b) en el exón 13 del gen ATP7B. La frecuencia alélica de los polimorfismos p. K832R y p.T991T en 27 pacientes cubanos con diagnóstico clínico de la Enfermedad de Wilson es 35,2 por ciento y 5,6 por ciento respectivamente. Conclusiones: Se analizó por primera vez en Cuba la combinación de los polimorfismos p. K832R y p. T991T que posibilitará hacer estudios moleculares por métodos indirectos(AU)

Introduction: Wilson's disease is characterized by accumulation of copper in the liver, brain and cornea. It is transmitted with an autosomal recessive inherited disorder. The molecular causes are mutations in the ATP7B gene. It has been reported in the literature more than 139polymorphisms of the ATP7B gene. Objective: Identify the conformational changes in exons 10 and 13 and detect the polymorphisms p.K832R and p.T991T in the ATP7B gene in Cuban patients with clinical diagnosis of Wilson's disease. Material and Methods: Was performed a descriptive study including 27 patients with Wilson’s disease ranging in the time from 2012 to 2013. Were applied the polymerase chain reaction to amplify the fragment of interest and the Conformation Polymorphism Single-Chain procedures in the exon 10 and 13 of the ATP7B gene. The p. K832R and p. T991T polymorphisms were detected by sequencing this fragment. Results: Three different conformational changes were identified: (a, b and c) in exon 10 and (a and b) in exon 13 of the ATP7B gene. The allelic frequency of polymorphisms p. K832R and p. T991T in 27 Cuban patients with clinical diagnosis of Wilson's disease is 35.2 percent and 5.6 percent, respectively. Conclusions: It is the first time in Cuba that a combination of the polymorphisms p. K832R and p. T991T were identified which will allow to make possible molecular studies by indirect methods(AU)

Detection of four DNA genetic marker systems to differentiate individuals in mixed seminal stain of two individuals / 中国法医学杂志

Objective To investigate personal identification of mixed seminal stain of two individuals, we combined the detection of genotyping autosomal, Y and X STR and sequencing mtDNA hypervariable Ⅰ (HV Ⅰ ) region. Methods We analyzed autosomal, Y and X STR with commercial kit and separating and sequencing HVⅠfragments of mixed seminal stain from two males by SSCP electrophoresis. Results Four genetic markers of the high amount sample can be obtained when mixed ratio is more than 1:10. When the proportion of two samples is close, the suspect could be excluded or, to some extent, identified by comparing with our results. Conclusion The combined detection of four genetic marker systems can, to some degree, solve the personal identification from mixed seminal stain of two individuals.

Bacterial ecology of abattoir wastewater treated by an anaerobic digestor

Abstract Wastewater from an anaerobic treatment plant at a slaughterhouse was analysed to determine the bacterial biodiversity present. Molecular analysis of the anaerobic sludge obtained from the treatment plant showed significant diversity, as 27 different phyla were identified. Firmicutes, Proteobacteria, Bacteroidetes, Thermotogae, Euryarchaeota (methanogens), and msbl6 (candidate division) were the dominant phyla of the anaerobic treatment plant and represented 21.7%, 18.5%, 11.5%, 9.4%, 8.9%, and 8.8% of the total bacteria identified, respectively. The dominant bacteria isolated were Clostridium, Bacteroides, Desulfobulbus, Desulfomicrobium, Desulfovibrio and Desulfotomaculum. Our results revealed the presence of new species, genera and families of microorganisms. The most interesting strains were characterised. Three new bacteria involved in anaerobic digestion of abattoir wastewater were published.

MOLECULAR VARIABILITY OF THREE GENES OF POTATO VEIN YELLOW VIRUS INFECTING Solanum tuberosum, USING SINGLE STRAND CONFORMATIONAL POLYMORPHISM / Variabilidad molecular de tres genes del virus del amarillamiento de las venas de la hoja de la papa que infecta Solanum tuberosum, mediante la técnica de polimorfismo conformacional de cadena sencilla

Potato yellow vein virus (PYVV), a virus with tripartite RNA (ss+) genome is classified as member of the genus Crinivirus within the family Closteoviridae. PYVV is the causal agent of potato yellow vein disease (PYVD) with yield loss between 25 %-50 % in field. Single strand conformational polymorphism (SSCP) has been reported to estimate variability in different viruses' species. In this study, the molecular variability of PYVV analyzed by SSCP patterns of three genes: major capsid protein (CP), minor capsid protein (CPm), and heat shock protein (Hsp70) obtained from 60 virus isolates from potato plants expressing PYVD. Leaves collected in Nariño, Colombia from 30 Solanum tuberosum Phureja Group (PhG) and 30 from the Andigena Group (AG). Genes amplified by RT-PCR and the purified PCR products used for SSCP. Three SSCP patterns detected for the CP gene, 12 for CPm and 12 for Hsp70. The pattern C was the most frequent for the Hsp70 gene and the pattern IV (33.3 %) for CPm gene in the Andigena Group. The pattern 1 for CP gene was present in 93.3 % of both host groups, indicating low number of variants compared to the CPm and Hsp70 genes. This is the first attempt to estimate intra e inter PYVV variability by the use of a simple molecular method considering three genes in a large group of potato samples affected by PYVD. SSCP technique was useful to evaluate the viral variability.

Potato yellow vein virus o virus del amarillamiento de venas de la hoja de la papa (PYVV) es un virus RNA tripartito (ss+) de la familia Closteroviridae género Crinivirus que causa la enfermedad de amarillamiento de nervaduras de la hoja de papa (PYVD) reduciendo la productividad, entre el 25 % y 50 %. La técnica de polimorfismo conformacional de cadena sencilla (SSCP) ha sido usada para estimar la variabilidad en diferentes especies de virus. En el presente trabajo se analizó la variabilidad molecular de 60 aislados de PYVV a partir de la comparación de tres genes: el gen de la proteína mayor de la cápside (CP), proteína menor de la cápside (CPm) y la proteína de choque térmico (Hsp70). Los aislados se obtuvieron de dos grupos de Solanum tuberosum: 30 del Grupo Phureja (GPh) y 30 del Grupo Andígena (GA), provenientes del departamento de Nariño, Colombia. Los genes se amplificaron por RT-PCR y los productos purificados se emplearon para SSCP en geles de poliacrilamida. Se detectaron tres perfiles de SSCP para el gen CP, 12 para el CPm y 12 para el Hsp70. Para el GA el perfil más frecuente del gen Hsp70 fue el perfil C (66,6 %) y para el gen CPm fue el IV (33,3 %). Para el gen CP, el patrón uno se encontró en el 93,3 % de los aislados, indicando menor variabilidad. Esta es la primera estimación de variabilidad de PYVV en diferentes genes y a través de una técnica molecular simple.

BRCA1 microsatellite instability in endometrial carcinoma and its relationship with clinical pathology / 实用医学杂志

Objective To study the occurrence of BRCA1 MSI in endometrial cancer and its relationship with clinic pathologic features; to explore the correlation between MSI and protein expression in BRCA1 gene. Methods Application of PCR-SSCP and DNA sequence analysis method was used to study D17S579 and D17S1349 in 49 sporadic endometrial cancer tissues, 20 cases with endometrial atypical hyperplasia and 28 cases with normal endometrial tissues. Results In the total samples of D17S579 and D17S1349, the three groups were significantly different: 34.69%(17/49) in the endometrial cancer group, 10%(2/20) in the endometrial atypical hyperplasia group and 7.14%(2/28) in the normal endometrium group (χ2= 11.208, P = 0.004). BRCA1 MSI positive rate related to the pathology grade and clinical stage, but no relationship was found in muscular infiltration depth, lymph node metastasis and histopathology types. In the endometrial cancer group, BRCA1 MSI positive rate and BRCA1 protein expression were in moderate correlated negatively (r = -0.779, P = 0.000). Conclusion BRCA1 MSI might play a role in the development of endometrial cancer, and low expression of BRCA1 protein. BRCA1 MSI might be associated with pathology grade and clinical stages in EC.

Trans Sialidase Genes Allow Clustering of TcI Trypanosoma cruzi Mexican Isolates.

Aims: Polymorphism of the trans sialidase (TS) family of genes is common in Trypanosoma cruzi. Our goal was to cluster Mexican TcI DTU (Discrete Typing Unit) using a set of primers specific for TS genes. Methodology: The DNA of 12 Mexican T. cruzi stocks (TcI) and reference strain were amplified using the CRP-1 primer, which anneals to the conserved 5' ends of CRP (Complement Regulatory Protein), TS, and FL-160 genes, and the CRP-2 primer, which anneals to conserved region within the GPI (Glycosil Phosphatidil Inositol) anchor sequence. Amplicons were analysed using PCR-SSCP (Single Strand Conformation Polymorphims) followed by construction of a nominal matrix data (presence/absence bands) to calculated the Jaccard and Dice similarity coefficient, and clustering with UPGMA. Results: Mexican TcI stocks produced a common pattern of amplification products and cluster in a separate group to CL-Brener strain (TcVI). The PCR-SSCP revealed that within the TcI Mexican stocks there were a complex pattern, but T. cruzi from the Yucatan peninsula clustered in special and separate group. Conclusion: The CRP-1 and CRP-2 primers were helpful for the analysis of genetic traits in T. cruzi DTU I and revealed the existence of special group in Yucatan Mexico.

Molecular genotyping of ABO blood groups in some population groups from India.

Background & objectives: Indian population is characterized by the presence of various castes and tribal groups. Various genetic polymorphisms have been used to differentiate among these groups. Amongst these, the ABO blood group system has been extensively studied. There is no information on molecular genotyping of ABO blood groups from India. Therefore, the main objective of this study was to characterize the common A, B and O alleles by molecular analysis in some Indian population groups. Methods: One hundred samples from the mixed population from Mumbai, 101 samples from the Dhodia tribe and 100 samples from the Parsi community were included in this study. Initially, the samples were phenotyped by standard serologic techniques. PCR followed by single strand conformational polymorphsim (SSCP) was used for molecular ABO genotyping. Samples showing atypical SSCP patterns were further analysed by DNA sequencing to characterize rare alleles. Results: Seven common ABO alleles with 19 different genotypes were found in the mixed population. The Dhodias showed 12 different ABO genotypes and the Parsis revealed 15 different ABO genotypes with six common ABO alleles identified in each of them. Two rare alleles were also identified. Interpretation & conclusions: This study reports the distribution of molecular genotypes of ABO alleles among some population groups from India. Considering the extremely heterogeneous nature of the Indian population, in terms of various genotype markers like blood groups, red cell enzymes, etc., many more ABO alleles are likely to be encountered.

Rapid detection of MDR-Mycobacterium tuberculosis using modified PCR-SSCP from clinical Specimens

<p><b>OBJECTIVE</b>To design a rapid test to detect the rifampin (RIF) and isoniazid (INH) resistant mutant based on polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) technique that analyzes the katG, rpoB genes.</p><p><b>METHODS</b>Biochemical test as well as IS6110 targeting PCR revealed 103 clinical samples were tuberculosis. To determine the susceptibility of isolates to anti TB drugs, the proportional method was used. Mutations presented within the amplified products of the katG, rpoB genes were evaluated by SSCP.</p><p><b>RESULTS</b>Using proportional method, 12 (11.6%) and 9 (8.7%) isolates were resistant respectively to INH and RIF and 9 (8.7%) isolates showed resistance to both drug (multi-drug resistant tuberculosis). Three (2.9%) multi-drug resistant tuberculosis and two INH resistant isolates were detected by the PCR-SSCP and sequencing. The sensitivity and specificity of PCR-SSCP for multi-drug resistant isolates were 33% and 100%, respectively.</p><p><b>CONCLUSIONS</b>Complete agreement between SSCP and sequencing can indicate that resistance-associated mutations have occurred in other genes except our considered genes.</p>

Rapid detection of MDR-Mycobacterium tuberculosis using modified PCR-SSCP from clinical Specimens

Objective: To design a rapid test to detect the rifampin (RIF) and isoniazid (INH) resistant mutant based on polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) technique that analyzes the katG, rpoB genes.Methods:tuberculosis. To determine the susceptibility of isolates to anti TB drugs, the proportional method was used. Mutations presented within the amplified products of the katG, rpoB genes were evaluated by SSCP.Results:Using proportional method, 12 (11.6%) and 9 (8.7%) isolates were resistant respectively Biochemical test as well as IS6110 targeting PCR revealed 103 clinical samples were to INH and RIF and 9 (8.7%) isolates showed resistance to both drug (multi-drug resistant tuberculosis). Three (2.9%) multi-drug resistant tuberculosis and two INH resistant isolates were detected by the PCR-SSCP and sequencing. The sensitivity and specificity of PCR-SSCP for multi-drug resistant isolates were 33% and 100%, respectively.Conclusions:Complete agreement between SSCP and sequencing can indicate that resistance-associated mutations have occurred in other genes except our considered genes.

Fingerprint study of Perns et Testis Cervi mtDNA with PCR-SSCP / 中国药学杂志

OBJECTIVE: To establish a method of molecular biology to identify the authenticity and varieties of Penis et Testis Cervi rapidly, simply and accurately. METHODS: The mitochondrial DNAs (mtDNA) of dried Cervus nippon Penis, Cervus elaphus. L Penis, Penis et Testis Bovis and commercial products were extracted and purified with column chromatography. A pair of primers were designed for PCR special amplification of mtDNA according to cytochrome b gene sequence in the cervidae mtDNA GenBank; the products of PCR were analyzed for the single strand conformation polymorphism (SSCP) by denaturing polyacrylamide gel electrophoresis (PAGE). RESULTS: The positive control samples and some commercial products showed clear single strand DNA (ssDNA) bands with different mobility, but the negative control sample and some other commercial products showed nothing. CONCLUSION: Column chromatography method is a good way for getting mtDNA with high purity and less destruction of structure for the follow-up special PCR amplification; SSCP technique not only shows ssDNA bands of mutually complementing clearly, but also reveals the difference of mobility among ssDNAs from all samples directly. For that reason, the technique of PCR-SSCP will be reliable and feasible to indentify the authenticity and varieties of products from cervidae.

Screening for Caveolin-3 gene polymorphism in Chinese Han diabetic patients / 实用医学杂志

Objective To observe the difference of caveolin-3(CAV3) gene polymorphism between normal people and diabetic patients in Chinese Han population. Methods Exon gene polymorphism in 50 normal people and 50 T2DM patients were detected by PCR-SSCP. Results The cumulative incidence rate of electrophoretic variation in T2DM patients was 48%, while cumulative incidence rate of normal people was 7%(P<0.001). It was proved that in the variant bands, there were base variant. Conclusions The variant base number of CAV3 gene in human T2DM samples are significantly more than the normal which can be preliminary detected by PCR-SSCP. It indicates that CAV3 gene polymorphism may be one of the genetic backgrounds for the occurence of Chinese T2DM.

Estudio de variantes intra-tipo del virus del papiloma humano tipo16, por análisis nucleotídico de la región MY09-MY11 / Study of intra-type variants of papillomavirus human tipo16, by analysis of the nucleotide region MY09-MY11

OBJETIVO: Identificar variantes intratipo de VPH16 mediante el análisis de la región MY09/ MY11 del gen L1, en el Laboratorio de Biología y Medicina Experimental Labiomex, Universidad de Los Andes. Mérida. MÉTODOS: Estudio descriptivo de corte transversal donde se procesaron 45 muestras del área genital, 38 femeninas y 7 masculinas, que presentaron infección por VPH16. Se realizó un análisis polimórfico de cadena sencilla del ADN (SSCP) con la finalidad de descubrir diferencias polimórficas de intratipo, se analizaron los diferentes polimorfismos hallados por secuenciación automática, y se establecieron las relaciones filogenéticas y funcionales de las variantes encontradas. RESULTADOS: Se logró detectar tres variantes intratipo de VPH16 de la región MY09/ MY11 del gen L1. Las variantes corresponden a sustituciones nucleotídicas sinónimas. Se determinó que las 3 variantes detectadas de VPH16 pertenecen a la clase europea. CONCLUSIONES: El análisis polimórfico de la región L1 permitió determinar la presencia de variabilidad intratipo en el gen L1 de VPH16. Todas las variaciones fueron de tipo mutación puntual, no se detectaron inserciones ni deleciones. Se logró identificar la secuencia del clon referencial VPH16, lo cual resulta importante para el desarrollo de sistemas de diagnóstico, tipificación y vacunas.

OBJECTIVE: Identify variants of HPV-16 intratiping by analyzing the region MY09 / MY11 L1 gene, en el Laboratory of Experimental Biology and Medicine LABIOMEX, Universidad de Los Andes. Merida. METHODS: Cross sectional study in which 45 samples were processed in the genital area, 38 female and 7 male, with infection by HPV16. Polymorphic analysis was performed on single stranded DNA (SSCP) in order to discover intratipo polymorphic differences were analyzed different polymorphisms found by automatic sequencing and phylogenetic relationships were established and functional variants found. RESULTS: Intratipo able to detect three variants of HPV16 in the study population in the region MY09 / MY11 L1 gene. Variants correspond to nucleotide substitutions synonymous. It was determined that 3 of HPV16 variants detected belong to the European class. CONCLUSION: The analysis of the polymorphic region MY09/ MY11 allowed determining the presence of intratipe variability in the HPV16 L1 gene. All variations were kind of mutation, there were no insertions or deletions. We identified the sequences of HPV-16 reference clone, which is important for the development of diagnostic systems, characterization and vaccines.

Identificación del polimorfismo k832r en pacientes cubanos con diagnóstico clínico de la enfermedad de Wilson / Identification of the k832r polymorphism in patients with clinical diagnosis of Wilson's disease

Introducción: la enfermedad de Wilson se caracteriza por la acumulación de cobre fundamentalmente en el hígado. Se transmite con un patrón de herencia autosómico recesivo. La causa molecular que la provoca son las mutaciones en el gen atp7b. Se han informado en la literatura varios polimorfismos en el gen atp7b. Objetivo: identificar el polimorfismo K832R en 100 pacientes cubanos diagnosticados clínicamente con la Enfermedad de Wilson. Material y Métodos: en el presente estudio se empleó la técnica de cribaje: Polimorfismo Conformacional de Simple Cadena para la determinación de cambios conformacionales en el exón 10. Se utilizó la Técnica de Secuenciación para la identificación del polimorfismo K832R. Resultados: se detectaron tres cambios conformacionales diferentes denominados: a, b y c. El cambio conformacional b y c correspondió al polimorfismo K832R en estado heterocigótico y homocigótico respectivamente. La frecuencia alélica del polimorfismo K832R en 100 pacientes cubanos diagnosticados clínicamente con la Enfermedad de Wilson es de 35%. Conclusiones: se identificó por primera vez en Cuba el polimorfismo K832R y posibilitará hacer estudios moleculares por métodos indirectos.

ABSTRACT Introduction: Wilson's disease is characterized by accumulation of copper in liver, brain and cornea. It is an autosomal recessive inherited disorder of copper metabolism. The molecular causes are mutations in the atp7b gene. It has been reported in the literature several polymorphisms in the atp7b gene. Objective: this research aims to identify the polymorphism K832R in 100 Cubans patients with clinical diagnosis of Wilson's disease. Materials and Methods: in this study we used the technique of screening: single stranded conformational polymorphism for the determination of conformational shifts in exon 10. We used sequencing technique for identifying the K832R polymorphism. Results: they identified three different conformational shifts denominated: a, b and c. The shifts b and c corresponded to polymorphism K832R in heterozygous and homozygous state respectively. The frequency of this polymorphism K832R is 35% in 100 Cubans patients. Conclusions: the polymorphism K832R was identified first in Cuba and it will make possible molecular studies by indirect methods.

Identification of Clematis species by PCR-SSCP analysis / 中国药学杂志

OBJECTIVE: To establish a rapid and accurate identification method to distinguish species of Clematis. METHODS: First, the registered ITS sequences of Clematis were seeked in the genbank database and compared with those of three species of Clematis' to confirm the stable specific identification site. Second, specific primers were designed, and PCR-SSCP was used to test Clematis. RESULTS: The fingerprints of the samples existed significant differences, indicating that our method was able to tell apart the different species of Clematis. CONCLUSION: PCR-SSCP has the advantages of high specificity and good reproducibility, therefore, it lays the foundation for the further development of accurate identification of Clematis.

Identificación de la mutación R579X en el exón 18 del gen RB1, en pacientes con lesiones cervicales asociadas a infección por virus del papiloma humano / Identification of the R579X mutation in exon 18 of the RB1 gene in patients with cervical infection associated with human papilloma virus

Identificar la mutación R579X ubicada en el exón 18 gen RB1, en pacientes con lesiones cervicales asociadas a infección por virus de papiloma humano mediante PCR-SSCP, PCR-RFLP y secuenciamiento. Estudio de 72 muestras de hisopado/cepillado de cuello uterino provenientes de 36 mujeres sanas, y 36 con infección por virus de papiloma humano y con presencia de lesiones cervicales a las cuales se les realizó la extracción de ADN, la amplificación del exón 18 del gen RB1 mediante PCR, y el análisis mediante PCR-SSCP, Secuenciamiento y PCR-RFLP. El análisis por PCR-SSCP reveló dos patrones diferentes de corrida y mediante el secuenciamiento se logró identificar la mutación R579X en el exón 18 del gen supresor de tumores RB1 en el 30,56 por ciento de las muestras experimentales. La mutación R579X encontrada en las muestras experimentales conlleva a la formación de una proteína truncada no funcional, y aunado a esto, el virus de papiloma humano podría estar favoreciendo a la inmortalización de las células que presentan dicha mutación

To identify the mutation R579X in exon 18 located RB1 gene in patients with cervical lesions associated with human papilloma virus infection by PCR-SSCP, PCR-RFLP and sequencing. Study of 72 samples of cervical brushing of the healthy women, infected with human papilloma virus and with cervical lesions that required DNA extraction, PCR amplification of exon 18 of the RB1 gene, PCR-SSCP, Sequencing and PCR-RFLP analysis. PCR-SSCP analysis revealed two different band patterns. We indentified the R579X mutation in exon 18 of the RB1 tumor suppressor gene in 30.56 percent of the experimental samples. The mutation found in the experimental samples leads to the formation of a non-functional truncated protein, in adition, human papilloma virus infection might contributed to the immortalization of cells with this mutation

Allelic frequency of the Kappa-Casein gene in Colombian and creole cattle breeds / Frecuencia alélica del gen Kappa-Caseína en bovinos criollos y colombianos / Freqüência do alelo de gene de Kappa-Caseína em bovinos crioulos e colombianos

Colombia has one of the most genetically diverse creole cattle populations, with eight creole breeds and two improved creole (Colombian) breeds. A high demand for meat and milk has led to the inevitable selection of highly productive cattle and the introduction of foreign breeds. Unfortunately, these breeds are often ill-suited for tropical conditions. These factors threaten the size of the creole livestock population, which is considered part of Colombia's national heritage. Objective: to estimate the allelic frequencies of the Kappa-Casein gene (CNS3) in Colombian creole cattle breeds (GCC). Methods: a total of 354 blood samples were taken from 30 animals of each of the following breeds: Blanco Orejinegro (BON), Caqueteño (CQT), Casanareño (CAS), Horned Costeño (Costeño con Cuernos, CCC), Chino Santandereano (ChS), Hartón del Valle (HV), Romosinuano (ROM), and Sanmartinero (SM), each representing the 8 established ''criollo'' (creole) breeds; the Lucerna (LUC) and Velasquez (VEL) representing the two Colombian improved breeds; and Brahman and Holstein as control breeds. DNA was extracted by a salting-out procedure and a 453 bp fragment on chromosome 6 was amplified by PCR. CSN3 alleles were identified using single strand conformation polymorphism (SSCP) and their sequence compared with those of the Genebank for Bos taurus and Bos indicus. Results: higher frequencies for allele variants of CSN3 A (0.39) and B (0.41) were found relative to the frequencies of I (0.038), G (0.095), A1 (0.025), E (0.006), and N (0.006). The allele of interest (CSN3 B) had a high frequency in the CCC (0.81), ROMO (0.66), CQT (0.55), ChS (0.48), and VEL (0.43) breeds. Conclusions: these findings suggest that Colombian creole breeds harbor a high genetic diversity which enriches its gene pool and warrants future conservation efforts to protect its integrity.

Colombia es uno de los países más diversos en recursos genéticos criollos. Posee ochos razas de ganado criollo (GCC) y dos razas de criollo mejorado o razas colombianas. La creciente demanda de alimentos ha generado una forzosa selección de individuos altamente productivos e introducción de razas foráneas (Holstein y Brahman) poco adaptadas a condiciones tropicales, lo que ha puesto en riesgo el tamaño efectivo del ganado criollo, considerado patrimonio nacional. Objetivo: estimar la frecuencia alélica del gen (CNS3) de la Kappa-Caseína en el (GCC). Métodos: se usaron 354 muestras de sangre de ocho razas bovinas criollas (30 individuos por raza): Blanco Orejinegro (BON), Caqueteño (CQT), Casanareño (CAS), Costeño con Cuernos (CCC), Chino Santandereano (ChS), Hartón del Valle (HV), Romosinuano (ROMO) y Sanmartinero (SM), dos colombianas Lucerna (LUC) y Velásquez (VEL) y dos controles Brahman y Holstein. Con el fin de estimar la frecuencia de los alelos k-caseína (k-CN) se amplificó un fragmento de 453 pb para k-CN (cromosoma 6). Los alelos se identificaron mediante la técnica PCR-SSCP. Resultados: se encontró mayor frecuencia para las variantes de k-CN A (0.39) y B (0.41), en comparación a I (0.038), G (0.095), A1 (0.025), E (0.006) y N (0.006). El alelo de interés k-CN B presentó alta frecuencia en las razas CCC (0.81), ROMO (0.66), CQT (0.55), ChS (0.48), y VEL (0.43). Conclusiones: la alta frecuencia del alelo de interés del gen k-CN ratifica al GCC como alternativa viable en esquemas sostenibles de producción de leche de mejor calidad y corrobora la necesidad de evaluación y caracterización de recursos zoogenético, como primer paso para su conservación.

A Colômbia é um dos países mais diversificado em recursos genéticos crioulos, tem oito bovinos da raça nativa e duas raças melhoradas ou raças crioulo colombiano (GCC). A alta demanda por alimentos tem levado a uma seleção forçada das pessoas altamente produtivas e/ou introdução de raças estrangeiras mal adaptados às condições tropicais, que têm prejudicado o tamanho efetivo de animais considerados património crioulo. Objetivo: estimar a frequência do alelo do gene Kappa-Caseína (CNS3) na (GCC). Métodos: foram utilizados 354 amostras de sangue de oito raças nativas (30 indivíduos por raça): Blanco Orejinegro (BON), Caqueteño (CQT), Casanareño (CAS), Costeño con Cuernos (CCC), Chino Santandereano (ChS), Hartón del Valle (HV), Romosinuano (ROM) e Sanmartinero (SM), dois Colombianas Lucerna (LUC) e Velásquez (VEL) e dois controles (Brahman and Holstein). Para estimar a freqüência de alelos de κ-caseína (CSN3), amplificaram um fragmento de 453 pb para CSN3. Os alelos foram identificados por PCR-SSCP. Resultados: encontramos uma maior freqüência de variantes do CSN3 A (0.39) e B (0.41), comparado com I (0.038), G (0.095), A1 (0.025), E (0.006) e N (0.006). O alelo de interesse CSN3 B apresentou alta freqüência em raças CCC (0.81), ROMO (0.66), CQT (0.55), CHS (0.48) e VEL (0.43). Conclusões: estes resultados sugerem que o CCG é um recurso genético, que abriga uma grande diversidade genética e apoia a necessidade de avaliação e caracterização dos recursos genéticos animais como um primeiro passo para a conservação.

Drug resistance of Mycobacterium tuberculosis strains in southern Brazil / Estudo da resistência de cepas de Mycobacterium tuberculosis aos antimicrobianos no sul do Brasil

INTRODUCTION: The aim of this work was to evaluate the prevalence of Mycobacterium tuberculosis (MT) strains with mutations that could result in resistance to the main drugs used in treatment in a region with one of the highest numbers of tuberculosis (TB) cases in southern Brazil. METHODS: Deoxyribonucleic acid (DNA) from 120 sputum samples from different patients suspicious of pulmonary tuberculosis who attended the Municipal Public Laboratory for Mycobacterium sp. diagnosis was directly amplified and analyzed by PCR-SSCP. The DNA was amplified in known hotspot mutation regions of the genes rpoB, ahpC, embB, katG, inhA, and pncA. RESULTS: The percentage of samples positive by culture was 9.2 percent (11/120); 5 percent (6/120) were positive by bacilloscopy and MT-PCR, and DNA fragments of the aforementioned resistance genes could be amplified from seven (7) of the eleven (11) samples with positive results, either by culture or PCR/bacilloscopy. All presented a SSCP pattern similar to a native, nonresistant genotype, with the ATCC strain 25177 as control, except for one sample (0.01 percent), which presented a SSCP profile demonstrating mutation at the embB gene. CONCLUSIONS: These results are consistent with the empirical observations by physicians treating TB patients in our region of a low occurrence of cases that are refractory to conventional treatment schemes, in contrast to other parts of the country. Continued surveillance, especially molecular, is essential to detect and monitor the outbreak of MT-resistant strains.

INTRODUÇÃO: O objetivo deste trabalho foi avaliar a prevalência de cepas de Mycobacterium tuberculosis (MT) com mutações que podem resultar em resistência às principais drogas utilizadas no tratamento em uma das regiões com o maior número de casos de tuberculose (TB) no Sul do Brasil. MÉTODOS: O ácido desoxiribonucleico (DNA) de 120 amostras de escarro de diferentes pacientes com suspeita de TB pulmonar que procuraram o serviço público de saúde do município sede da região para o diagnóstico de MT foi diretamente amplificado e analisado por PCR-SSCP. Foram amplificadas regiões conhecidas onde ocorrem a maioria das mutações nos genes rpoB, ahpC, embB, katG, inhA, and pncA. RESULTADOS: Nove virgula dois por cento (11/120) das amostras apresentaram resultado positivo por cultura, 5 por cento (6/120) foi positiva por bacilscopia e PCR para MT, e os fragmentos dos genes mencionados puderam ser amplificados em sete (7) dos onze (11) casos com resultado positivo, seja por cultura ou PCR/baciloscopia. Todos estes casos apresentaram um padrão de SSCP similar ao genótipo nativo, não resistente, por comparação com a cepa controle ATCC 25177, com exceção de uma amostra (0,01 por cento), que apresentou um padrão de SSCP mutante no gene embB. CONCLUSÕES: Estes resultados são consistentes com as observações empíricas por parte dos clínicos que tratam os pacientes com TB na região, de uma baixa ocorrência de casos refratários ao tratamento convencional, em contraste com outras partes do país. Porém, a vigilância contínua, especialmente molecular, é essencial para identificar e monitorar o aparecimento de cepas de MT resistentes.