Página 12 dos resultados de 264 itens digitais encontrados em 0.008 segundos

‣ Efficient bicistronic expression of cre in mammalian cells.

Gorski, J A; Jones, K R
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/05/1999 Português
Relevância na Pesquisa
27.854238%
Cre recombinase-mediated DNA recombination is proving to be a powerful technique for the generation of mosaic mutant mice. To develop this technology further, we have altered the cre gene to enhance its expression in mammalian cells and have tested its efficiency of expression in a bicistronic message. Using a transient transfection assay, we found that the extension of a eukaryotic translation initiation consensus sequence, the insertion of two N-terminal amino acids, and the mutation of a cryptic splice acceptor site did not detectably alter Cre recombinase activity. The addition of either of two introns resulted in an approximately 2-fold increase in recombination frequency. We then tested the relative efficacy of Cre-mediated recombination in several bicistronic messages having the encephalomyocarditis virus internal ribosome entry site (IRES). Recombination frequencies were only reduced 2-fold relative to a comparable monocistronic cre gene. The latter results indicate that it will be possible to generate transgenic mouse strains having tissue-specific expression of the Cre recombinase through integration of an IRES-cre gene without disabling the targeted gene.

‣ Genomewide Trapping of Genes that Encode Secreted and Transmembrane Proteins Repressed by Oncogenic Signaling

Gebauer, Mathias; von Melchner, Harald; Beckers, Thomas
Fonte: Cold Spring Harbor Laboratory Press Publicador: Cold Spring Harbor Laboratory Press
Tipo: Artigo de Revista Científica
Publicado em /11/2001 Português
Relevância na Pesquisa
27.64385%
A retroviral gene trap containing a human CD2 cell surface antigen/neomycin–phosphotransferase fusion gene in the U3 region of its LTR (U3Ceo) was used to screen the mammalian genome for genes encoding secreted and/or transmembrane proteins that are repressed by oncogenic transformation. From an integration library consisting of cells transformable by the insulin-like growth factor 1 (IGF-1), a collection of neomycin resistant (NeoR) clones was obtained; 86% also expressed the CD2 cell surface antigen. Molecular analysis of a random sample of NeoR clones revealed that the U3Ceo gene trap preferentially disrupted genes coding for secreted and transmembrane proteins. In each case, the signal sequence of the endogenous gene was fused in-frame to the CD2/neomycin–phosphotransferase reporter gene due to a cryptic splice acceptor site embedded in the coding region of the CD2 cDNA. When the library was transformed by IGF-1 and selected against CD2 expression, integrations were obtained in genes that are repressed by transformation. Molecular analysis of six randomly chosen integrations revealed that, in each case, U3Ceo captured a signal sequence from proteins involved in oncogenic transformation and metastatic spread.

‣ Oncogenic point mutations in exon 20 of the RB1 gene in families showing incomplete penetrance and mild expression of the retinoblastoma phenotype.

Onadim, Z; Hogg, A; Baird, P N; Cowell, J K
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/07/1992 Português
Relevância na Pesquisa
27.64385%
The retinoblastoma-predisposition gene, RB1, segregates as an autosomal dominant trait with high (90%) penetrance. Certain families, however, show an unusual low-penetrance phenotype with many individuals being unaffected, unilaterally affected, or with evidence of spontaneously regressed tumors. We have used single-strand conformation polymorphism analysis and PCR sequencing to study two such families. Mutations were found in exon 20 of RB1 in both cases. In one family a C----T transition in codon 661 converts an arginine (CGG) to a tryptophan (TGG) codon. In this family, incomplete penetrance and mild phenotypic expression were observed in virtually all patients, possibly indicating that single amino acid changes may modify protein structure/function such that tumorigenesis is not inevitable. In the second family the mutation in codon 675 is a G----T transversion that converts a glutamine (GAA) to a stop (TAA) codon. However, this mutation also occurs near a potential cryptic splice acceptor site, raising the possibility of alternative splicing resulting in a less severely disrupted protein.

‣ A 57-Nucleotide Upstream Early Polyadenylation Element in Human Papillomavirus Type 16 Interacts with hFip1, CstF-64, hnRNP C1/C2, and Polypyrimidine Tract Binding Protein

Zhao, Xiaomin; Öberg, Daniel; Rush, Margaret; Fay, Joanna; Lambkin, Helen; Schwartz, Stefan
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /04/2005 Português
Relevância na Pesquisa
27.64385%
We have investigated the role of the human papillomavirus type 16 (HPV-16) early untranslated region (3′ UTR) in HPV-16 gene expression. We found that deletion of the early 3′ UTR reduced the utilization of the early polyadenylation signal and, as a consequence, resulted in read-through into the late region and production of late L1 and L2 mRNAs. Deletion of the U-rich 3′ half of the early 3′ UTR had a similar effect, demonstrating that the 57-nucleotide U-rich region acted as an enhancing upstream element on the early polyadenylation signal. In accordance with this, the newly identified hFip1 protein, which has been shown to enhance polyadenylation through U-rich upstream elements, interacted specifically with the HPV-16 upstream element. This upstream element also interacted specifically with CstF-64, hnRNP C1/C2, and polypyrimidine tract binding protein, suggesting that these factors were either enhancing or regulating polyadenylation at the HPV-16 early polyadenylation signal. Mutational inactivation of the early polyadenylation signal also resulted in increased late mRNA production. However, the effect was reduced by the activation of upstream cryptic polyadenylation signals, demonstrating the presence of additional strong RNA elements downstream of the early polyadenylation signal that direct cleavage and polyadenylation to this region of the HPV-16 genome. In addition...

‣ A new alternative transcript encodes a 60 kDa truncated form of integrin beta 3.

Djaffar, I; Chen, Y P; Creminon, C; Maclouf, J; Cieutat, A M; Gayet, O; Rosa, J P
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 15/05/1994 Português
Relevância na Pesquisa
27.64385%
A cDNA for integrin beta 3 isolated from a human erythroleukaemia (HEL) cell library contained a 340 bp insert at position 1281. This mRNA, termed beta 3c, results from the use of a cryptic AG donor splice site in intron 8 of the beta 3 gene, and is different from a previously described alternative beta 3 mRNA. The predicted open reading frame of beta 3C stops at a TAG stop codon 69 bp downstream from position 1281. It starts with the signal peptide and the 404 N-terminal extracellular residues of beta 3, encompassing the ligand binding sites, followed by 23 C-terminal intron-derived residues, corresponding to a truncated form of beta 3 lacking the cysteine-rich, transmembrane and cytoplasmic domains. Expression of beta 3C mRNA was demonstrated in human platelets, megakaryocytes, endothelial cells and HEL cells by reverse transcriptase/PCR. The beta 3C transcript was also demonstrated in the mouse, suggesting its conservation through evolution. Finally, a 60 kDa polypeptide corresponding to the beta 3C alternative transcript was demonstrated in platelets by Western blotting using a polyclonal antibody raised against a synthetic peptide designed from the beta 3C intronic sequence. Taken together, these results suggest a biological role for beta 3C...

‣ Inhibiting farnesylation of progerin prevents the characteristic nuclear blebbing of Hutchinson-Gilford progeria syndrome

Capell, Brian C.; Erdos, Michael R.; Madigan, James P.; Fiordalisi, James J.; Varga, Renee; Conneely, Karen N.; Gordon, Leslie B.; Der, Channing J.; Cox, Adrienne D.; Collins, Francis S.
Fonte: National Academy of Sciences Publicador: National Academy of Sciences
Tipo: Artigo de Revista Científica
Português
Relevância na Pesquisa
27.854238%
Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disorder that is characterized by dramatic premature aging and accelerated cardiovascular disease. HGPS is almost always caused by a de novo point mutation in the lamin A gene (LMNA) that activates a cryptic splice donor site, producing a truncated mutant protein termed “progerin.” WT prelamin A is anchored to the nuclear envelope by a farnesyl isoprenoid lipid. Cleavage of the terminal 15 aa and the farnesyl group releases mature lamin A from this tether. In contrast, this cleavage site is deleted in progerin. We hypothesized that retention of the farnesyl group causes progerin to become permanently anchored in the nuclear membrane, disrupting proper nuclear scaffolding and causing the characteristic nuclear blebbing seen in HGPS cells. Also, we hypothesized that blocking farnesylation would decrease progerin toxicity. To test this hypothesis, the terminal CSIM sequence in progerin was mutated to SSIM, a sequence that cannot be farnesylated. SSIM progerin relocalized from the nuclear periphery into nucleoplasmic aggregates and produced no nuclear blebbing. Also, blocking farnesylation of authentic progerin in transiently transfected HeLa, HEK 293, and NIH 3T3 cells with farnesyltransferase inhibitors (FTIs) restored normal nuclear architecture. Last...

‣ Expression of novel isoforms of carnitine palmitoyltransferase I (CPT-1) generated by alternative splicing of the CPT-ibeta gene.

Yu, G S; Lu, Y C; Gulick, T
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 15/08/1998 Português
Relevância na Pesquisa
27.64385%
Carnitine palmitoyltransferase I (CPT-I) catalyses the rate-determining step in mitochondrial fatty acid beta-oxidation. The enzyme has two cognate structural genes that are preferentially expressed in liver (alpha) or fat and muscle (beta). We hypothesized the existence of additional isoforms in heart to account for unique kinetic characteristics of enzyme activity in this tissue. Hybridization and PCR screening of a human cardiac cDNA library revealed the expression of two novel CPT-I isoforms generated by alternative splicing of the CPT-Ibeta transcript, in addition to the beta and alpha cDNA species previously described. Ribonuclease protection and reverse transcriptase-mediated PCR assays confirmed the presence of mRNA species of each splicing variant in heart, skeletal muscle and liver, with differing relative concentrations in the tissues. The novel splicing variants omit exons or utilize a cryptic splice donor site within an exon. Deduced polypeptide sequences of the novel enzymes include omissions in the region of putative membrane-spanning and malonyl-CoA regulatory domains compared with the previously described CPT-Is, implying that the encoded enzymes will exhibit unique features with respect to outer mitochondrial membrane topology and response to physiological and pharmacological inhibitors.

‣ Functionally null mutations in patients with the cblG-variant form of methionine synthase deficiency.

Wilson, A; Leclerc, D; Saberi, F; Campeau, E; Hwang, H Y; Shane, B; Phillips, J A; Rosenblatt, D S; Gravel, R A
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /08/1998 Português
Relevância na Pesquisa
27.64385%
Methionine synthase (MS) catalyses the methylation of homocysteine to methionine and requires the vitamin B12 derivative, methylcobalamin, as cofactor. We and others have recently cloned cDNAs for MS and described mutations associated with the cblG complementation group that correspond to MS deficiency. A subset of cblG, known as "cblG variant," shows no detectable MS activity and failure of [57Co]CN cobalamin to incorporate into MS in patient fibroblasts. We report the mutations responsible for three cblG-variant patients, two of them siblings, who presented with neonatal seizures, severe developmental delay, and elevated plasma homocysteine. Cell lines from all three patients were negative by northern blotting, though trace MS mRNA could be detected by means of phosphorimage analysis. Reverse transcriptase-PCR, SSCP, and nucleotide sequence analysis revealed four mutations. All were functionally null, creating either a frameshift with a downstream stop codon or an insert containing an internal stop codon. Of the two mutations found in the siblings, one of them, intervening sequence (IVS)-166A-->G, generates a cryptic donor splice site at position -166 of an intron beginning after Leu113, resulting in a 165-bp insertion of intronic sequence at junction 339/340. The second is a 2-bp deletion...

‣ Progressive vascular smooth muscle cell defects in a mouse model of Hutchinson–Gilford progeria syndrome

Varga, Renee; Eriksson, Maria; Erdos, Michael R.; Olive, Michelle; Harten, Ingrid; Kolodgie, Frank; Capell, Brian C.; Cheng, Jun; Faddah, Dina; Perkins, Stacie; Avallone, Hedwig; San, Hong; Qu, Xuan; Ganesh, Santhi; Gordon, Leslie B.; Virmani, Renu; Wight
Fonte: National Academy of Sciences Publicador: National Academy of Sciences
Tipo: Artigo de Revista Científica
Português
Relevância na Pesquisa
27.64385%
Children with Hutchinson–Gilford progeria syndrome (HGPS) suffer from dramatic acceleration of some symptoms associated with normal aging, most notably cardiovascular disease that eventually leads to death from myocardial infarction and/or stroke usually in their second decade of life. For the vast majority of cases, a de novo point mutation in the lamin A (LMNA) gene is the cause of HGPS. This missense mutation creates a cryptic splice donor site that produces a mutant lamin A protein, termed “progerin,” which carries a 50-aa deletion near its C terminus. We have created a mouse model for progeria by generating transgenics carrying a human bacterial artificial chromosome that harbors the common HGPS mutation. These mice develop progressive loss of vascular smooth muscle cells in the medial layer of large arteries, in a pattern very similar to that seen in children with HGPS. This mouse model should prove valuable for testing experimental therapies for this devastating disorder and for exploring cardiovascular disease in general.

‣ Reciprocal regulation of glycine-rich RNA-binding proteins via an interlocked feedback loop coupling alternative splicing to nonsense-mediated decay in Arabidopsis

Schöning, Jan C.; Streitner, Corinna; Meyer, Irmtraud M.; Gao, Yahong; Staiger, Dorothee
Fonte: Oxford University Press Publicador: Oxford University Press
Tipo: Artigo de Revista Científica
Português
Relevância na Pesquisa
27.64385%
The Arabidopsis RNA-binding protein AtGRP8 undergoes negative autoregulation at the post-transcriptional level. An elevated AtGRP8 protein level promotes the use of a cryptic 5′ splice site to generate an alternatively spliced transcript, as_AtGRP8, retaining the 5′ half of the intron with a premature termination codon. In mutants defective in nonsense-mediated decay (NMD) abundance of as_AtGRP8 but not its pre-mRNA is elevated, indicating that as_AtGRP8 is a direct NMD target, thus limiting the production of functional AtGRP8 protein. In addition to its own pre-mRNA, AtGRP8 negatively regulates the AtGRP7 transcript through promoting the formation of the equivalent alternatively spliced as_AtGRP7 transcript, leading to a decrease in AtGRP7 abundance. Recombinant AtGRP8 binds to its own and the AtGRP7 pre-mRNA, suggesting that this interaction is relevant for the splicing decision in vivo. AtGRP7 itself is part of a negative autoregulatory circuit that influences circadian oscillations of its own and the AtGRP8 transcript through alternative splicing linked to NMD. Thus, we identify an interlocked feedback loop through which two RNA-binding proteins autoregulate and reciprocally crossregulate by coupling unproductive splicing to NMD. A high degree of evolutionary sequence conservation in the introns retained in as_AtGRP8 or as_AtGRP7 points to an important function of these sequences.

‣ Epidermal expression of the truncated prelamin A causing Hutchinson–Gilford progeria syndrome: effects on keratinocytes, hair and skin

Wang, Yuexia; Panteleyev, Andrey A.; Owens, David M.; Djabali, Karima; Stewart, Colin L.; Worman, Howard J.
Fonte: Oxford University Press Publicador: Oxford University Press
Tipo: Artigo de Revista Científica
Português
Relevância na Pesquisa
27.64385%
Hutchinson–Gilford progeria syndrome (HGPS) is an accelerated aging disorder caused by point mutation in LMNA encoding A-type nuclear lamins. The mutations in LMNA activate a cryptic splice donor site, resulting in expression of a truncated, prenylated prelamin A called progerin. Expression of progerin leads to alterations in nuclear morphology, which may underlie pathology in HGPS. We generated transgenic mice expressing progerin in epidermis under control of a keratin 14 promoter. The mice had severe abnormalities in morphology of skin keratinocyte nuclei, including nuclear envelope lobulation and decreased nuclear circularity not present in transgenic mice expressing wild-type human lamin A. Primary keratinocytes isolated from these mice had a higher frequency of nuclei with abnormal shape compared to those from transgenic mice expressing wild-type human lamin A. Treatment with a farnesyltransferase inhibitor significantly improved nuclear shape abnormalities and induced the formation of intranuclear foci in the primary keratinocytes expressing progerin. Similarly, spontaneous immortalization of progerin-expressing cultured keratinocytes selected for cells with normal nuclear morphology. Despite morphological alterations in keratinocyte nuclei...

‣ Intragenic deletions and a deep intronic mutation affecting pre-mRNA splicing in the dihydropyrimidine dehydrogenase gene as novel mechanisms causing 5-fluorouracil toxicity

van Kuilenburg, André B. P.; Meijer, Judith; Mul, Adri N. P. M.; Meinsma, Rutger; Schmid, Veronika; Dobritzsch, Doreen; Hennekam, Raoul C. M.; Mannens, Marcel M. A. M.; Kiechle, Marion; Etienne-Grimaldi, Marie-Christine; Klümpen, Heinz-Josef; Maring, Ja
Fonte: Springer-Verlag Publicador: Springer-Verlag
Tipo: Artigo de Revista Científica
Português
Relevância na Pesquisa
27.64385%
Dihydropyrimidine dehydrogenase (DPD) is the initial enzyme acting in the catabolism of the widely used antineoplastic agent 5-fluorouracil (5FU). DPD deficiency is known to cause a potentially lethal toxicity following administration of 5FU. Here, we report novel genetic mechanisms underlying DPD deficiency in patients presenting with grade III/IV 5FU-associated toxicity. In one patient a genomic DPYD deletion of exons 21–23 was observed. In five patients a deep intronic mutation c.1129–5923C>G was identified creating a cryptic splice donor site. As a consequence, a 44 bp fragment corresponding to nucleotides c.1129–5967 to c.1129–5924 of intron 10 was inserted in the mature DPD mRNA. The deleterious c.1129–5923C>G mutation proved to be in cis with three intronic polymorphisms (c.483 + 18G>A, c.959–51T>G, c.680 + 139G>A) and the synonymous mutation c.1236G>A of a previously identified haplotype. Retrospective analysis of 203 cancer patients showed that the c.1129–5923C>G mutation was significantly enriched in patients with severe 5FU-associated toxicity (9.1%) compared to patients without toxicity (2.2%). In addition, a high prevalence was observed for the c.1129–5923C>G mutation in the normal Dutch (2.6%) and German (3.3%) population. Our study demonstrates that a genomic deletion affecting DPYD and a deep intronic mutation affecting pre-mRNA splicing can cause severe 5FU-associated toxicity. We conclude that screening for DPD deficiency should include a search for genomic rearrangements and aberrant splicing.

‣ Identification of a MIP mutation that activates a cryptic acceptor splice site in the 3′ untranslated region

Jin, Chongfei; Jiang, Jin; Wang, Wei; Yao, Ke
Fonte: Molecular Vision Publicador: Molecular Vision
Tipo: Artigo de Revista Científica
Publicado em 02/11/2010 Português
Relevância na Pesquisa
27.64385%

‣ CYP17A1 Intron Mutation Causing Cryptic Splicing in 17α-Hydroxylase Deficiency

Hwang, Daw-Yang; Hung, Chi-Chih; Riepe, Felix G.; Auchus, Richard J.; Kulle, Alexandra E.; Holterhus, Paul-Martin; Chao, Mei-Chyn; Kuo, Mei-Chuan; Hwang, Shang-Jyh; Chen, Hung-Chun
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 26/09/2011 Português
Relevância na Pesquisa
27.64385%
17α-hydroxylase/17, 20-lyase deficiency (17OHD) is an autosomal recessive disease causing congenital adrenal hyperplasia and a rare cause of hypertension with hypokalemia. The CYP17A1 gene mutation leads to 17OHD and its clinical features. We described an 18 y/o female with clinical features of 17α-hydroxylase/17, 20-lyase deficiency and characterized the functional consequences of an intronic CYP17A1 mutation. The coding regions and flanking intronic bases of the CYP17A1 gene were amplified by PCR and sequenced. The patient is a compound heterozygote for the previously described p.R358X and IVS1 +2T>C mutations. A first intron splice donor site mutation was re-created in minigene and full-length expression vectors. Pre-mRNA splicing of the variant CYP17A1 intron was studied in transfected cells and in a transformed lymphoblastoid cell line. When the full-length CYP17A1 gene and minigene containing the intronic mutation was expressed in transfected cells, the majority (>90%) of mRNA transcripts were incorrectly spliced. Only the p.R358X transcript was detected in the EBV-transformed lymphoblastoid cell line. The IVS1 +2T>C mutation abolished most 17α-hydroxylase/17, 20-lyase enzyme activity by aberrant mRNA splicing to an intronic pseudo-exon...

‣ Progerin and telomere dysfunction collaborate to trigger cellular senescence in normal human fibroblasts

Cao, Kan; Blair, Cecilia D.; Faddah, Dina A.; Kieckhaefer, Julia E.; Olive, Michelle; Erdos, Michael R.; Nabel, Elizabeth G.; Collins, Francis S.
Fonte: American Society for Clinical Investigation Publicador: American Society for Clinical Investigation
Tipo: Artigo de Revista Científica
Português
Relevância na Pesquisa
27.64385%
Hutchinson-Gilford progeria syndrome (HGPS), a devastating premature aging disease, is caused by a point mutation in the lamin A gene (LMNA). This mutation constitutively activates a cryptic splice donor site, resulting in a mutant lamin A protein known as progerin. Recent studies have demonstrated that progerin is also produced at low levels in normal human cells and tissues. However, the cause-and-effect relationship between normal aging and progerin production in normal individuals has not yet been determined. In this study, we have shown in normal human fibroblasts that progressive telomere damage during cellular senescence plays a causative role in activating progerin production. Progressive telomere damage was also found to lead to extensive changes in alternative splicing in multiple other genes. Interestingly, elevated progerin production was not seen during cellular senescence that does not entail telomere shortening. Taken together, our results suggest a synergistic relationship between telomere dysfunction and progerin production during the induction of cell senescence, providing mechanistic insight into how progerin may participate in the normal aging process.

‣ Mutation identification in a canine model of X-linked ectodermal dysplasia

Casal, Margret L.; Scheidt, Jennifer L.; Rhodes, James L.; Henthorn, Paula S.; Werner, Petra
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /07/2005 Português
Relevância na Pesquisa
27.96444%
X-linked hypohidrotic ectodermal dysplasia (XHED), an inherited disease recognized in humans, mice, and cattle, is characterized by hypotrichosis, a reduced number or absence of sweat glands, and missing or malformed teeth. In a subset of affected individuals and animals, mutations in the EDA gene (formerly EDI), coding for ectodysplasin, have been found to cause this phenotype. Ectodysplasin is a homotrimeric transmembrane protein with an extracellular TNF-like domain, which has been shown to be involved in the morphogenesis of hair follicles and tooth buds during fetal development. Some human XHED patients also have concurrent immunodeficiency, due to mutations in the NF-κB essential modulator protein (IKBKG; formerly NEMO), which is also encoded on the X chromosome. In a breeding colony of dogs with XHED, immune system defects had been suspected because of frequent pulmonary infections and unexpected deaths resulting from pneumonia. To determine if defects in EDA or IKBKG cause XHED in the dogs, linkage analysis and sequencing experiments were performed. A polymorphic marker near the canine EDA gene showed significant linkage to XHED. The canine EDA gene was sequenced and a nucleotide substitution (G to A) in the splice acceptor site of intron 8 was detected in affected dogs. In the presence of the A residue...

‣ Deciphering intrafamilial phenotypic variability by exome sequencing in a Bardet–Biedl family

González-del Pozo, María; Méndez-Vidal, Cristina; Santoyo-Lopez, Javier; Vela-Boza, Alicia; Bravo-Gil, Nereida; Rueda, Antonio; García-Alonso, Luz; Vázquez-Marouschek, Carmen; Dopazo, Joaquín; Borrego, Salud; Antiñolo, Guillermo
Fonte: Wiley Periodicals, Inc. Publicador: Wiley Periodicals, Inc.
Tipo: Artigo de Revista Científica
Português
Relevância na Pesquisa
27.854238%
Bardet–Biedl syndrome (BBS) is a model ciliopathy characterized by a wide range of clinical variability. The heterogeneity of this condition is reflected in the number of underlying gene defects and the epistatic interactions between the proteins encoded. BBS is generally inherited in an autosomal recessive trait. However, in some families, mutations across different loci interact to modulate the expressivity of the phenotype. In order to investigate the magnitude of epistasis in one BBS family with remarkable intrafamilial phenotypic variability, we designed an exome sequencing–based approach using SOLID 5500xl platform. This strategy allowed the reliable detection of the primary causal mutations in our family consisting of two novel compound heterozygous mutations in McKusick–Kaufman syndrome (MKKS) gene (p.D90G and p.V396F). Additionally, exome sequencing enabled the detection of one novel heterozygous NPHP4 variant which is predicted to activate a cryptic acceptor splice site and is only present in the most severely affected patient. Here, we provide an exome sequencing analysis of a BBS family and show the potential utility of this tool, in combination with network analysis, to detect disease-causing mutations and second-site modifiers. Our data demonstrate how next-generation sequencing (NGS) can facilitate the dissection of epistatic phenomena...

‣ Molecular basis of the cell-surface expression of immunoglobulin mu chain without light chain in human B lymphocytes.

Pollok, B A; Anker, R; Eldridge, P; Hendershot, L; Levitt, D
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /12/1987 Português
Relevância na Pesquisa
27.854238%
Four distinct human B-lymphoid cell lines possess the ability to circumvent the mechanism regulating intracellular transport of immunoglobulin protein. These cells do not produce light chains, yet they express mu heavy chains on the cell surface at comparable levels to B-cell lines that produce native forms of both proteins. The mu-chain mRNA produced in all four cell lines was found to contain an identical deletion of most of the heavy-chain variable (VH) region (75% of the 3' portion), with no apparent alteration in constant (C) region structure. The truncated mu (mu*)-chain mRNA in these cells was created through the use of a cryptic splice donor site found within the human VH gene(s) utilized by these B-cell lines. The truncated mu chains exhibited a decreased ability to associate with the intracellular transport regulatory protein, heavy-chain binding protein (BiP). This result indicates that VH region structure, in addition to C mu 1 region structure, influences the formation of the BiP recognition site on the heavy chain. Furthermore, it suggests that the mechanism allowing for cell-surface expression of the mu* chains in the absence of light-chain pairing is the inability of BiP to bind to the mu* chains and hence prevent their intracellular transport. The high frequency with which the mu-only surface immunoglobulin positive phenotype is present in our collection of human B-cell lines and the isolation of one of the cell lines from a healthy individual also suggest that B cells of this type may represent a significant subpopulation among the normal human B-cell repertoire.

‣ Improved vectors for stable expression of foreign genes in mammalian cells by use of the untranslated leader sequence from EMC virus.

Kaufman, R J; Davies, M V; Wasley, L C; Michnick, D
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 25/08/1991 Português
Relevância na Pesquisa
27.854238%
Dicistronic mRNA expression vectors efficiently translate a 5' open reading frame (ORF) and contain a selectable marker within the 3' end which is inefficiently translated. In these vectors, the efficiency of translation of the selectable 3' ORF is reduced approximately 100-fold and is highly dependent on the particular sequences inserted into the 5' cloning site. Upon selection for expression of the selection marker gene product, deletions within the 5' ORF occur to yield more efficient translation of the selectable marker. We have generated improved dicistronic mRNA expression vectors by utilization of a putative internal ribosomal entry site isolated from encephalomyocarditis (EMC) virus. Insertion of the EMC virus leader sequence upstream of an ORF encoding either a wildtype or methotrexate resistant dihydrofolate reductase (DHFR) reduces DHFR translation up to 10-fold in a monocistronic DHFR expression vector. However, insertion of another ORF upstream of the EMC leader to produce a dicistronic mRNA does not further reduce DHFR translation. In the presence of the EMC virus leader, DHFR translation is not dependent on sequences inserted into the 5' end of the mRNA. We demonstrate that stable high level expression of inserted cDNAs may be rapidly achieved by selection for methotrexate resistance in DHFR deficient as well as DHFR containing cells. In contrast to previously described dicistronic expression vectors...

‣ The Drosophila suppressor of sable protein binds to RNA and associates with a subset of polytene chromosome bands.

Murray, M V; Turnage, M A; Williamson, K J; Steinhauer, W R; Searles, L L
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /04/1997 Português
Relevância na Pesquisa
27.854238%
Mutations of the Drosophila melanogaster suppressor of sable [su(s)] gene, which encodes a 150-kDa nuclear protein [Su(s)], increase the accumulation of specific transcripts in a manner that is not well understood but that appears to involve pre-mRNA processing. Here, we report biochemical analysis of purified, recombinant Su(s) [rSu(s)] expressed in baculovirus and in Escherichia coli as maltose binding protein (MBP) fusions and immunocytochemical analysis of endogenous Su(s). This work has shown that purified, baculovirus-expressed rSu(s) binds to RNA in vitro with a high affinity and limited specificity. Systematic evolution of ligands by exponential enrichment was used to identify preferred RNA targets of rSu(s), and a large proportion of RNAs isolated contain a full or partial match to the consensus sequence UCAGUAGUCU, which was confirmed to be a high-affinity rSu(s) binding site. An MBP-Su(s) fusion protein containing the N-terminal third of Su(s) binds RNAs containing this sequence with a higher specificity than full-length, baculovirus-expressed rSu(s). The consensus sequence resembles both a cryptic 5' splice site and a sequence that is found near the 5' end of some Drosophila transcripts. Immunolocalization studies showed that endogenous Su(s) is distributed in a reticulated pattern in Drosophila embryo and salivary gland nuclei. In salivary gland cells...