Progress in transcriptional studies / 生物工程学报

Gene expression exhibits temporal and spatial patterns to response environmental changes and growth cycle. Gene expression is under strict control at different levels among which control at transcription level is the predominant mode, especially in prokaryotes. In this review, we summarized the new developments of methods used in transcriptional studies, including modifications and improvements of the classic methods, such as gel-shift assay, DNA foot printing, and in vivo reporter system. In addition, we introduced examples to apply new methods, such as surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) to characterize protein-DNA, ligand-protein, and ligand-protein-DNA interactions. The collection of these methods and their application could guide and accelerate relevant studies.

[Conventional Vs. reverse nerve grafting for peripheral nerve repair in lower extremity of rats]

Autograft is the best option in nerve defects when end-to-end repair can not sufficiently preserve nerve continuity. Theoretically, if the severed nerve is reversely grafted, it may prevent axonal growth into nerve branches, and larger amounts of axons will reach the target organ and more satisfactory results will be obtained. In this study we aimed to compare conventional versus reverse nerve grafting. This study was performed in Animal laboratory of Hazrat Fatemeh Hospital from April till August 2011. We randomly divided 40 Wistar rats into two groups. We excised 1.5 cm of the right sciatic nerve and anastomosed it conventionally between the proximal and distal ends of the nerve in rats in group A and in a reverse manner in rats in group B. The rats' footprints were recorded in the first and 16[th] weeks after surgery. In week 16, the grafted nerves were removed under anesthesia for pathological examination and axon count. Subsequently, the results were compared clinically by sciatic functional index [SFI] through footprint analysis and paraclinically by axon count. A p-value smaller than 0.05 was considered statistically significant. Conventional and reverse nerve grafting no had statistically significant differences in clinical assessment in the first and 16[th] weeks [P=0.87] post-surgically and also no difference in paraclinical assessment in week 16 [P=0.68]. We had no significant clinically or para clinically differences between two approaches. It should be considered that the diameter and length of nerves and muscles in human is larger than rats, so the results of nerve repair may differ in human. We suggest a study in animal model which is anatomically more similar to human

Frecuencias alélicas de 14 STR’s autosómicos en una población de Antioquia, Colombia / Allele frequencies of 14 STR’s autosomal in a population of Antioquia, Colombia

En esta investigación se analizaron 50 individuos independientes de una muestra total de 5´671.689 habitantes correspondientes al Departamento de Antioquia en las ciudades de Medellín, Envigado, Sabaneta y Santa Fe de Antioquia, con el fin de realizar la huella genética. Los STR autosómicos utilizados fueron CSF1PO, TH01, TPOX, D16S539, D7S820, D13S317, F13A01, vWA, HPRTB, D8S1179, D5S818, PENTA E, D18S51 y D3S1358. Con los datos obtenidos de las frecuencias alélicas se analizó el equilibrio de Hardy–Weinberg, el índice de fijación y algunos parámetros forenses, mediante los programas Genepop Versión 3.2 y PowerState. Se observó que la población no presentó diferencias significativas para la prueba de Hardy – Weinberg y en su confirmación con la prueba de F. Los resultados médicos forenses mostraron un índice de discriminación acumulado de 0,999992947, el índice de exclusión estuvo por encima del 0,9906 y la probabilidad de coincidencia acumulada fue de 1 en 7,05311E-06 individuos.

Efficiency of RAPD and ISSR markers system in accessing genetic variation of rice bean (Vigna umbellata) landraces

Vigna umbellata (Thunb.) Ohwi and Ohashi commonly known as rice bean or climbing mountain bean is under-exploited tropical legume. Genetic variation between 10 landraces of rice bean was evaluated using random amplified polymorphic DNA (RAPD) and inter simple sequence repeats (ISSR) markers. Among these markers, RAPD primers generated 987 amplification products of which 719 were polymorphic and ISSR markers produced 479 amplification products, out of which 296 were polymorphic. RAPD fingerprinting detected more polymorphic loci (70.30 percent) than the ISSR fingerprinting (61.79 percent). Mean PIC (polymorphism information content) for each of these marker systems (0.243 for RAPD and 0.203 for ISSR) suggested that both the marker systems were equally effective in determining polymorphisms. The dendrograms constructed using RAPD and ISSR marker systems were highly correlated with each other as revealed by high Mantel correlation (r = 0.95). Pairwise similarity index values ranged from 0.530 to 0.782 (RAPD), 0.608 and 0.862 (ISSR) and 0.559 to 0.777 (RAPD and ISSR) and mean similarity index value of 0.677, 0.729 and 0.694 for RAPD, ISSR and combined data, respectively. RAPD and ISSR marker systems were found to be useful for the genetic diversity studies in V. umbellata and identify variation within landraces.

Advances in the chemical and biological studies of polyamides / 药学学报

Polyamides, containing N-methylpyrrole (Py) and N-methyl-imidazole (Im) amino acids, are synthetic oligomers programmed to read the DNA double helix in the minor groove with high affinities and sequence specificities resulting in modulation of gene expression. They are cell permeable, stable and have no cytotoxicity, which provide a promising tool of gene regulation. We describe here recent advances in the field of DNA binding polyamides, including pairing rules, specifities and affinities to DNA, synthesis methods, cellular and nuclear uptake properties, gene regulation and effectiveness in vivo. The potential problems and difficulties in future research are also discussed.

Mechanism of Castration-induced Apoptosis of Ventral Prostate in Rat / 대한내분비학회지

BACKGROUNDS: Castration-induced androgen deprivation triggers a sequence of events, which activates apoptotic cell death of the androgen-dependent epithelial cells within the rat ventral prostate. To investigate the mechanism of castration-dependent apoptosis in the rat ventral prostate, the regulation of apoptosis-related genes was been investigated. METHODS: Azaline B was subcutaneously injected into Sprague-Dawley rat. The Fas receptor (Fas), Fas ligand (FasL) and bcl-2 mRNA, as well as the protein levels were detected by RT-PCR and Western blot analyses. Azaline B-dependent apoptosis was determined using TUNEL and a DNA fragmentation assay. The transacting factor of the FasL promoter was identified by DNA footprinting and a DNA mobility shift assay. RESULTS: The rat prostate was regressed after castration, with and the involuted ventral prostate regenerated by testosterone pretreatment, but not by that with FSH. Apoptosis of the ventral prostate was detected, after castration, using toluidine blue staining, a TUNEL assay and an apoptotic DNA fragmentation assay. The levels of Fas, FasL mRNA and protein were increased after castration. In the DNase I footprinting assay, using the FasL promoter and a nuclear extract prepared from a control prostate, at least two sites were protected: the SP-1 binding site at -283 bp and the prostate-unidentified factor(P-UF) binding site at -247 bp. The SP-1 binding activity vanished in the nuclear extract prepared from castrated rats. In the DNA mobility shift assay, the SP-1 binding activity was slightly decreased after castration. Both the Bcl-2 mRNA and Bcl-2 protein were downregulated after castration. CONCLUSION: These results suggest that the Fas/FasL system and Bcl-2 may be important to castrationdependent apoptosis in the rat ventral prostate, with SP-1 related to the castration-dependent regulation of the FasL gene

Mechanism of Castration-induced Apoptosis of Ventral Prostate in Rat / 대한내분비학회지

BACKGROUNDS: Castration-induced androgen deprivation triggers a sequence of events, which activates apoptotic cell death of the androgen-dependent epithelial cells within the rat ventral prostate. To investigate the mechanism of castration-dependent apoptosis in the rat ventral prostate, the regulation of apoptosis-related genes was been investigated. METHODS: Azaline B was subcutaneously injected into Sprague-Dawley rat. The Fas receptor (Fas), Fas ligand (FasL) and bcl-2 mRNA, as well as the protein levels were detected by RT-PCR and Western blot analyses. Azaline B-dependent apoptosis was determined using TUNEL and a DNA fragmentation assay. The transacting factor of the FasL promoter was identified by DNA footprinting and a DNA mobility shift assay. RESULTS: The rat prostate was regressed after castration, with and the involuted ventral prostate regenerated by testosterone pretreatment, but not by that with FSH. Apoptosis of the ventral prostate was detected, after castration, using toluidine blue staining, a TUNEL assay and an apoptotic DNA fragmentation assay. The levels of Fas, FasL mRNA and protein were increased after castration. In the DNase I footprinting assay, using the FasL promoter and a nuclear extract prepared from a control prostate, at least two sites were protected: the SP-1 binding site at -283 bp and the prostate-unidentified factor(P-UF) binding site at -247 bp. The SP-1 binding activity vanished in the nuclear extract prepared from castrated rats. In the DNA mobility shift assay, the SP-1 binding activity was slightly decreased after castration. Both the Bcl-2 mRNA and Bcl-2 protein were downregulated after castration. CONCLUSION: These results suggest that the Fas/FasL system and Bcl-2 may be important to castrationdependent apoptosis in the rat ventral prostate, with SP-1 related to the castration-dependent regulation of the FasL gene

Mechanism of Azaline B-induced Apoptosis of Ventral Prostate in Rat / 대한비뇨기과학회지

PURPOSE: Androgen deprivation triggers a sequence of events that activates apoptotic cell death of the androgen-dependent epithelial cells within the rat ventral prostate, ultimately resulting in the involution of the gland. To investigate the mechanism of azaline B-dependent apoptosis in the rat ventral prostate, the regulation of apoptosis-related genes were examined. MATERIALS AND METHODS: Azaline B was subcutaneously injected in Sprague-Dawley rat. Fas receptor(Fas), Fas ligand(FasL), bcl-2 mRNA, and protein levels were detected by RT-PCR and Western blot. Azaline B-dependent apoptosis was determined by TUNEL and DNA fragmentation assay. Transacting factor of FasL promoter was identified by DNA footprinting and DNA mobility shift assay. RESULTS: The prostate regressed after azaline B treatment in rat, and the involuted ventral prostate regenerated after testosterone pretreatment. Apoptosis of the ventral prostate was detected by TUNEL assay and apoptotic DNA fragmentation assay after azaline B treatment. The levels of Fas and FasL mRNA and protein increased after azaline B treatment. In DNase I footprinting assay with FasL promoter using nuclear extract prepared from control prostate, at least two sites were protected: SP-1 binding site at -283bp and prostate-unidentified factor(P-UF) binding site at -247bp. SP-1 binding activity vanished in the nuclear extract prepared from azaline B-treated rats. In the DNA mobility shift assay, SP-1 binding activity decreased after azaline B treatment. Bcl-2 mRNA and protein were downregulated after azaline B treatment. CONCLUSIONS: These results suggest that Fas/FasL system and Bcl-2 are important to azaline B-dependent apoptosis in rat ventral prostate and that SP-1 is related to azaline B-dependent regulation of the FasL gene.

Polymorphisms and functions of the aldose reductase gene 5' regulatory region in Chinese patients with type 2 diabetes mellitus / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>To screen the 5' regulatory region of the aldose reductase (AR) gene for genetic variabilities causing changes in protein expression and affecting the promoter function.</p><p><b>METHODS</b>The screenings were carried out by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). All SSCP variants were submitted for DNA sequencing and inserted into the plasmid chloromycetin acetyl transferase (CAT) enhancer vector. The constructs were used to transfect Hela cells, and CAT assays were performed to assess promoter activity. Gel mobility shift and footprinting assays were also performed to determine the interaction between the DNA and nuclear proteins.</p><p><b>RESULTS</b>Two polymorphisms, C (-106) T and C (-12) G, were identified in the regulatory region in 123 Chinese control subjects and 145 patients with type 2 diabetes mellitus. The frequencies of genotypes WT/WT, WT/C (-12) G and WT/C (-106) T were not significantly different between the subjects and patients. In the patients with and without retinopathy, frequencies of WT/C (-106) T were 31.5% and 17.5% (P < 0.05) respectively, and the frequencies of WT/C (-12) G were 10.5% and 2.5% (P > 0.05) respectively. The total frequency of WT/C (-12) G and WT/C (-106) T in patients with retinopathy was 41.8%, significantly higher than that (20.0%) in patients without retinopathy (P < 0.025). The relative transcription activities of the wild-type, the C (-12) G and the C (-106) T were 15.7%, 31.0% and 32.2%, respectively. The results of DNA-protein interaction assays showed that these variations did not change the binding site of DNA with trans-acting factors.</p><p><b>CONCLUSION</b>The polymorphisms C (-12) G and C (-106) T strongly associated with diabetic retinopathy in the Chinese population have been identified in the regulatory region of the aldose reductase gene.</p>

La huella genética del DNA en Varones: haplotipo del cromosom Y en una población mexicana analizando cinco STRs / Male DNA profile: Y-chromosome haplotypes in a Mexican population analyzing five STRs

Antecedentes: Las repeticiones cortas en tándem o S TR s (short tandem repeats) localizadas en la región no seudo-autosómica del cromosoma Y , son marcadores moleculares utilizados para obtener la huella genética del DNA específicamente en varones, lo cual permite resolver casos especiales en el campo de la Medicina Legal. Analizando varios S TR s para formar haplotipos del cromosoma Y , se pueden solucionar de manera sencilla pruebas de paternidad donde el supuesto padre no está disponible, así como situaciones forenses, como casos de violación donde se encuentran mezclas de DNA de hombre y mujer. Métodos: Cinco STRs del cromosoma Y recientemente informados: A4, A7.1, A7.2, A10 y C4 (White et al. 1999) fueron tipificados en 101 mestizos mexicanos del Noroeste de México mediante PCR, electroforesis en gel de poliacrilamida y tinción de plata. Resultados: Se estimaron frecuencias alélicas de cada S TR . El rango de diversidad genética de estos marcadores fue de 57.1 por ciento para A-4 a 74.7 por ciento para C-4. Con excepción de A-4, las distribuciones alélicas de los cinco S TR s fueron similares (p>0.05) a la del reporte original. Se observaron 75 haplotipos diferentes de los 98 haplotipos completos obtenidos. Este sistema de cinco S TR s presentó una diversidad haplotípica de 99.0 por ciento y una capacidad de discriminación de 77.5 por ciento (76/98) en la muestra poblacional estudiada. Conclusiones: Estos marcadores STRs del cromosoma Y representan un gran potencial para identificar varones y líneas paternas, y pueden usarse confiablemente para lograr exclusiones en pruebas forenses y de paternidad en población mexicana.

Medicina hoy: biología molecular y medicina a fines del siglo XX / Medicine today: molecular biology and medicine at the end of the 20th century

Este trabajo revisa conceptos de la biología molecular moderna con la premisa de que su influencia en la medicina de nuestros días es tan grande que ya no puede ser un saber limitado a unos pocos expertos. Se analiza la estructura y organización de los genes humanos, su naturaleza partida en exones e intrones y el flujo de información genética desde el DNA a la proteína. Se describe el papel del control de la transcripción en la regulación de la expresión genética y la diferenciación celular, presentando ejemplos de experimentos que definen la importancia de los genes "maestros". Se describen los conceptos básicos de la ingeniería genética, la generación de animales transgénicos y knock out y las aplicaciones de la biología molecular al diangóstico médico y a la determinación de identidad y lazos biológicos. Finalmente la terapia génica y las realidades y fantasías de eventuales transgénesis o clonado por trasplante nuclear en humanos.

Prueba de compatibilidad inmunogenética / Immunogenetic compatibility test

En este trabajo presentamos nuestra experiencia en la utilización de la prueba de compatibilidad inmunogenética para establecer vínculos biológicos en tríos típicos. En la década del 70 nuestro Servicio utilizaba únicamente los sistemas eritrocitarios que nos permitían excluir solamente el 20 por ciento de los hombres falsamente alegados como padres. Posteriormente incorporamos al estudio de los antígenos del sistema HLA, que posibilitó la inclusión del padre alegado como biológico, con probabilidades de paternidad comprendidas entre el 90 al 98 por ciento, según la frecuencia poblacional del halotipo HLA obligado. El desarrollo de las técnicas de biología molecular ha permitido incorporar a las pericias realizadas en nuestro laboratorio el análisis del polimorfismo del ADN. Proponemos que la prueba de compatibilidad inmunogenética comprenda el estudio de marcadores fenotípicos (antígenos de los sistemas eritrocitarios y del sistema HLA) y genotípicos (polimorfismo del ADN). La utilización de esta metodología permite la inclusión de la paternidad con una confiabilidad de los resultados superior al 99,99 por ciento.

Characterization of regulatory elements on the promoter region of human ATP-citrate lyase

ATP-citrate lyase (ACL), an enzyme catalyzing the first step in biosynthesis of fatty acids, is induced during the lipogenesis and cholesterologenesis. We demonstrate that the region -213 to -128 of human ACL promoter is responsible for conferring glucose-mediated transcription. This region in the ACL promoter contains Sp1 binding sites determined by DNase I foot-printing assay. Gel retardation assay using oligonucleotides from -179 to -141 and -140 to -110 showed two specific DNA-protein complexes postulated to be formed by transcription factor Sp1. Competition gel shift and supershift assays have confirmed that these DNA-protein complexes were the result of induced Sp1 as well as another Sp1-related proteins. Western blot analysis also demonstrated that transcription factor Sp1 was slightly increased in the nuclear proteins extracted from Alexander cells following supplementation of glucose. In addition, expression of 110 kDa protein reacting with antibody against Sp3 was dramatically increased by glucose supplementation, while isoforms of Sp3, about 80 kDa in size was decreased in its amounts. Our results suggest that changes in the expression of Sp1 family proteins play an important role in activation of the ACL promoter by glucose.

Identificación de restos óseos humanos mediante análisis de ADN / Identification of human bones remains using ADN analysis

En los últimos años la tecnología del ADN se ha convertido en una poderosa herramienta para la identificación individual, incluso cuando sólamente se cuenta con restos óseos. Se ilustra un caso en el cual se realizó la prueba de identificación comparando el ADN de los supuestos padres contra el ADN extraído del hueso de un menor de sexo femenino, utilizando los marcadores : HLA-DQA1, LDLR, GYPA, HBGG, D7S8, Gc, y D1S80. Los perfiles genéticos obtenidos permitieron concluir una identificación positiva de los restos como pertenecientes a la niña desaparecida. Esta metodología queda disponible para futuros casos forenses que así lo ameriten

A mechanism of differential expression of GLUT2 in hepatocyte and pancreatic beta-cell line

DNase I footprinting assay using liver nuclear extracts revealed six protected regions between nucleotide -600 and +110 and hence named Box I-VI. Upstream promoter element (UPE), a DNA element playing crucial role in transcriptional control of the tissue specific expression of pancreatic beta-cell, has been detected within the proximal region of rat GLUT2 promoter. This region is included in Box VI. The protein-DNA interaction in this region (Box VI) was confirmed by mobility shift assay using liver nuclear extracts. Deletion of the region between -585 bp and -146 bp resulted in dramatic changes in promoter activity when they were expressed in liver and beta-cell derived cell line. When -585/-146 construct was expressed in liver, the activity was decreased to 46%, whereas the activity in beta-cell line, HIT-T15 cell, was increased by 84% when compared to -146/+190 construct. These opposing phenomena can be explained by the fact that beta-cell specifically expresses the UPE binding protein. Assuming that there may be Box VI-binding protein playing negative roles both in hepatocyte and beta-cell, and that the protein acts as a negative regulator of GLUT2 gene, the UPE binding protein in the beta-cell may overcome the inhibition by binding to the protein.

Molecular analysis of the human fetal-to-adult globin switching.

Hereditary persistence of fetal hemoglobin (HPFH) is a condition characterized by continued expression of the gamma-globin gene in adult life. Analysis of a Japanese HPFH family had revealed that the C-T transition at position -114 within the distal CCAAT box of the gamma-globin gene associated with the HPFH allele. In the vicinity of the distal CCAAT box, other two mutations (-117 C-T, 13 bp del) had been identified in individuals with a HPFH phenotype. Functional analysis of these mutant promoters in erythroid cell lines suggested that the distal CCAAT box works positively in the fetus but negatively in the adult on the expression of the gamma-globin gene. Further study on transgenic mice showed that the -114 mutation was responsible for the elevated expression of the gamma-globin gene in the adult. To elucidate the molecular mechanism underlying the persistent expression of the gamma-globin genes associated with the HPFH mutations, interaction of the mutant promoters with nuclear factors was analyzed. Relevance of the nuclear factor, NFE3, to the gamma-globin regulation was suggested by the affected binding of NFE3 to the altered distal CCAAT boxes with HPFH mutations (-117, -114, 13 bp del).