Codon and mRNA sequence optimization of microdystrophin transgenes improves expression and physiological outcome in dystrophic mdx mice following AAV2/8 gene transfer

Duchenne muscular dystrophy is a fatal muscle-wasting disorder. Lack of dystrophin compromises the integrity of the sarcolemma and results in myofibers that are highly prone to contraction-induced injury. Recombinant adenoassociated virus (rAAV)-mediated dystrophin gene transfer strategies to muscle for the treatment of Duchenne muscular dystrophy (DMD) have been limited by the small cloning capacity of rAAV vectors and high titers necessary to achieve efficient systemic gene transfer. In this study, we assess the impact of codon optimization on microdystrophin (ΔAB/R3-R18/ΔCT) expression and function in the mdx mouse and compare the function of two different configurations of codon-optimized microdystrophin genes (ΔAB/R3-R18/ΔCT and ΔR4-R23/ΔCT) under the control of a muscle-restrictive promoter (Spc5-12). Codon optimization of microdystrophin significantly increases levels of microdystrophin mRNA and protein after intramuscular and systemic administration of plasmid DNA or rAAV2/8. Physiological assessment demonstrates that codon optimization of ΔAB/R3-R18/ΔCT results in significant improvement in specific force, but does not improve resistance to eccentric contractions compared with noncodon-optimized ΔAB/ R3-R18/ΔCT. However, codon-optimized microdystrophin ΔR4-R23/ΔCT completely restored specific force generation and provided substantial protection from contraction-induced injury. These results demonstrate that codon optimization of microdystrophin under the control of a muscle-specific promoter can significantly improve expression levels such that reduced titers of rAAV vectors will be required for efficient systemic administration.

Promoter analysis and immunolocalisation show that puroindoline genes are exclusively expressed in starchy endosperm cells of wheat grain

The purolindolines are small cysteine-rich proteins which are present in the grain of wheat. They have a major impact on the utilisation of the grain as they are the major determinants of grain texture, which affects both milling and baking properties. Bread and durum wheats were transformed with constructs comprising the promoter regions of the Puroindoline a (Pina) and Puroindoline b (Pinb) genes fused to the uidA (GUS) reporter gene. Nine lines showing 3:1 segregation for the transgene and comprising all transgene/species combinations were selected for detailed analysis of transgene expression during grain development. This showed that transgene expression occurred only in the starchy endosperm cells and was not observed in any other seed or vegetative tissues. The location of the puroindoline proteins in these cells was confirmed by tissue printing of developing grain, using a highly specific monoclonal antibody for detection and an antibody to the aleurone-localised 8S globulin as a control. This provides clear evidence that puroindolines are only synthesised and accumulated in the starchy endosperm cells of the wheat grain.

Characterisation of chicken riboflavin carrier protein gene structure and promoter regulation by estrogen

Estrogen is an important steroid hormone that mediates most of its effects on regulation of gene expression by binding to intracellular receptors. The consensus estrogen response element (ERE) is a 13 bp palindromic inverted repeat with a three nucleotide spacer. However, several reports suggest that many estrogen target genes are regulated by diverse elements, such as imperfect EREs and ERE half sites (ERE 1/2),which are either the proximal or the distal half of the palindrome. To gain more insight into ERE half site-mediated gene regulation, we used a region from the estrogen-regulated chicken riboflavin carrier protein (RCP) gene promoter that contains ERE half sites. Using moxestrol, an analogue of estrogen and transient transfection of deletion and mutation containing RCP promoter/reporter constructs in chicken hepatoma (LMH2A) cells, we identified an estrogen response unit (ERU) composed of two consensus ERE 1/2 sites and one non-consensus ERE 1/2 site. Mutation of any of these sites within this ERU abolishes moxestrol response. Further, the ERU is able to confer moxestrol responsiveness to a heterologous promoter. Interestingly, RCP promoter is regulated by moxestrol in estrogen responsive human MCF-7 cells, but not in other cell lines such as NIH3T3 and HepG2 despite estrogen receptor-alpha (ER-�) co transfection. Electrophoretic mobility shift assays (EMSAs) with promoter regions encompassing the half sites and nuclear extracts from LMH2A cells show the presence of a moxestrol-induced complex that is abolished by a polyclonal anti-ER� antibody. Surprisingly, estrogen receptor cannot bind to these promoter elements in isolation. Thus, there appears to be a definite requirement for some other factor(s) in addition to estrogen receptor, for the generation of a suitable response of this promoter to estrogen. Our studies therefore suggest a novel mechanism of gene regulation by estrogen, involving ERE half sites without direct binding of ER to the cognate elements.

Use of microprojectile bombardment in transient expression assays to analyze protochlorophyllide reductase gene expression in greening maize seedling leaf cells

In young cells of leaf meristems the progenitors of chloroplasts are small organelles known as proplastids, which divide and differentiate into chloroplasts. However, in the absence of light, proplastids undergo a different sequence of development and become etioplasts. When light is supplied to etiolated plants during the "greening" process, etioplasts differentiate into chloroplasts containing chlorophyll. An important light dependent step in chlorophyll biosynthesis is the photoreduction of protochlorophyllide to chlorophyllide by the NADPH:protochlorophyllide reductase (PCR) enzyme. This enzyme is present at high activity only in etiolated tissue and during early stages of light-induced chlorophyll synthesis. The enzyme and its corresponding mRNAs decrease dramatically with prolonged exposure to light. We have investigated the light-dependent transcriptional regulation of a PCR gene in greening maize leaf cells using a transient expression assay based on microprojectile bombardment. The promoter region was isolated and cloned into a ?-glucuronidase (GUS) reporter gene expression plasmid. We have used this chimeric plasmid in tungsten particle bombardment of both etiolated and greening maize seedling leaves to determine whether the cloned promoter region contains regulatory sequences that control light-responsive PCR gene expression.

Low levels of realized seed and pollen gene flow and strong spatial genetic structure in a small, isolated and fragmented population of the tropical tree Copaifera langsdorffii Desf

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Granulocytic sarcoma of the small intestine with CBF beta/MYH11 fusion gene: report of an aleukaemic case and review of the literature

Granulocytic sarcomas (GS) are rare extramedullary tumours composed of immature myeloid cells. Inversion of chromosome 16 [inv(16)] is a cytogenetic marker for M4Eo subtype of acute myeloid leukaemia (AML). The possibility of an association between the development of granulocytic sarcoma of the small intestine (GSSI) and the M4Eo subtype of AML was suggested in nine previous case reports.Here we report an aleukaemic case of GSSI with inv(16) and its molecular equivalent, the CBFbeta/MYH11 fusion gene, detected by reverse transcriptase-polymerase chain reaction (RT-PCR), that after treatment with conventional AML chemotherapy followed by autologous bone marrow transplantation, achieved complete haematological and molecular remission on bone marrow examination. After chemotherapy, a thickened ileum wall positive for CBFbeta/MYH11 on tumour mass samples was still observed on computed tomography (CT) studies, raising the question of residual GS representing a reservoir of malignant cells. This case demonstrates the critical need of multidisciplinary diagnosis and follow-up of this entity combining immunopathologic, cytogenetic and molecular studies, reinforcing the potentiality of risk-adapted therapy strategies, as it is increasingly claimed for patients with overt AML. (C) 2003 Elsevier Ltd. All rights reserved.

Characterization and transcriptional analysis of the promoter region of the Duffy blood group, chemokine receptor (DARC) gene in cattle

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

The promoter of a gene encoding an isoflavone reductase-like protein in coffee (Coffea arabica) drives a stress-responsive expression in leaves

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Haplotype effects on matrix metalloproteinase-1 gene promoter activity in cancer cells

Increased expression of matrix metalloproteinase-1 (MMP1) is associated with poor prognosis in cancers. Several single nucleotide polymorphisms (-1607GG > G, -839G > A, -755G > T, -519A > G, -422T > A, -340C > T, and 320C > T) in the MMP1 gene promoter have recently been identified. In this study, we assessed the functional effects of these polymorphisms on MMP1 gene promoter activity in cell lines of melanoma (A2058 and A375), breast cancer (MCF7 and MDA-MB-231), lung cancer (A549 and H69), and colorectal cancer (HT-29, SW-620) by comparing the promoter strengths of 10 most common haplotypes deriving from these polymorphisms. In A2058 cells, the GG-G-G-A-T-T-T and GG-G-G-A-C-T haplotypes had 2-fold higher promoter activity than the GG-G-T-A-T-T-C, GG-G-G-A-A-T-T, GG-G-G-A-T-T-C, and GG-G-G-A-A-C-T haplotypes, which in turn, had 3-fold higher promoter activity than the G-G-T-A-A-C-T, G-A-T-G-T-T-T, G-A-T-G-A-C-T, and G-A-T-G-A-T-G haplotypes. In A375 and MDA-MB-231 cells, high expression haplotypes include not only the -1607GG-bearing haplotypes but also the G-A-T-G-A-T-T haplotype containing the -1607G allele. A similar trend was detected in A549 cells. In addition, in A549 cells, the GG-G-G-A-T-T-T haplotype had > 2-fold higher promoter activity than several other 1607GG-bearing haplotypes. In MCF7 cells, the GG-G-G-A-T-T-T and G-G-T-A-A-C-T haplotypes had 1.5- to 4-fold higher promoter activity than the other haplotypes. These results suggest that the polymorphisms exert haplotype effects on the transcriptional regulation of the MMP1 gene in cancer cells, and indicate a need to examine haplotypes rather than any single polymorphism in genetic epidemiologic studies of the MMP1 gene in cancers.

cAMP signaling pathway controls glycogen metabolism in Neurospora crassa by regulating the glycogen synthase gene expression and phosphorylation

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

A systematic approach to identify STRE-binding proteins of the gsn glycogen synthase gene promoter in Neurospora crassa

The gene encoding glycogen synthase in Neurospora crassa (gsn) is transcriptionally down-regulated when mycelium is exposed to a heat shock from 30 to 45 degrees C. The gsn promoter has one stress response element (STRE) motif that is specifically bound by heat shock activated nuclear proteins. In this work, we used biochemical approaches together with mass spectrometric analysis to identify the proteins that bind to the STRE motif and could participate in the gsn transcription regulation during heat shock. Crude nuclear extract of heat-shocked mycelium was prepared and fractionated by affinity chromatography. The fractions exhibiting DNA-binding activity were identified by electrophoretic mobility shift assay (EMSA) using as probe a DNA fragment containing the STRE motif DNA-protein binding activity was confirmed by Southwestern analysis. The molecular mass (MM) of proteins was estimated by fractionating the crude nuclear extract by SDS-PAGE followed by EMSA analysis of the proteins corresponding to different MM intervals. Binding activity was detected at the 30-50 MM kDa interval. Fractionation of the crude nuclear proteins by IEF followed by EMSA analysis led to the identification of two active fractions belonging to the pIs intervals 3.54-4.08 and 6.77-7.31. The proteins comprising the MM and pI intervals previously identified were excised from a 2-DE gel, and subjected to mass spectrometric analysis (MALDI-TOF/TOF) after tryptic digestion. The proteins were identified by search against the MIPS and MIT N. crassa databases and five promising candidates were identified. Their structural characteristics and putative roles in the gsn transcription regulation are discussed.

Characterization of ndh gene of isoniazid resistant and susceptible Mycobacterium tuberculosis isolates from Brazil

Resistance in Mycobacterium tuberculosis to isoniazid (INH) is caused by mutations in the catalase-peroxidase gene (katG) , and within the inhA promoter and/or in structural gene. A small percentage (~ 10%) of INH-resistant strains do not present mutations in both of these loci. Other genes have been associated with INH resistance including the gene encoding for NADH dehydrogenase (ndh) . Here we report the detection of two ndh locus mutations (CGT to TGT change in codon 13 and GTG to GCG change in codon 18) by analyzing 23 INH-resistant and in none of 13 susceptible isolates from Brazilian tuberculosis patients. We also detected two isolates without a mutation in ndh, or any of the other INH resistance-associated loci examined, suggesting the existence of additional, as yet to be described, INH resistance mechanisms.

Epstein-barr virus infection and single nucleotide Polymorphisms in the promoter region of interleukin 10 gene in patients with Hodgkin lymphoma

Context. - Hodgkin lymphoma is a neoplastic disease in which the immune system plays a major role in its pathogenesis. Interleukin 10 ( IL-10), an immunosuppressive cytokine actively produced in patients with Hodgkin lymphomas, favors the survival of the Hodgkin/Reed-Sternberg cells. Individual variations in IL-10 levels may be due, in part, to the presence of single nucleotide polymorphisms in the IL10 gene promoter.Objective. - To evaluate whether particular single nucleotide polymorphisms in the IL10 gene are found more frequently in Hodgkin lymphoma cases associated with Epstein-Barr virus infection.Design. - the identification of single nucleotide polymorphisms at positions -1082 and -819/-592 in the IL10 gene was performed by polymerase chain reaction and restriction length fragment polymorphisms analysis in 65 cases of Hodgkin lymphoma and 50 cases of reactive benign follicular lymphoid hyperplasia ( non-Hodgkin lymphoma control group).Results. - the frequency of the genotype GG at position -1082 was found to be significantly higher in patients with Epstein-Barr virus-positive Hodgkin lymphoma compared with Epstein-Barr virus-negative cases.Conclusions. - the results suggest that the presence of specific single nucleotide polymorphisms in the IL10 gene, notably those associated with high IL-10 production, may play a role in the susceptibility to Epstein-Barr virus -positive Hodgkin lymphoma development.

Genomic organization of the Neurospora crassa gsn gene: possible involvement of the STRE and HSE elements in the modulation of transcription during heat shock

Glycogen synthase, an enzyme involved in glycogen biosynthesis, is regulated by phosphorylation and by the allosteric ligand glucose-6-phosphate (G6P). In addition, enzyme levels can be regulated by changes in gene expression. We recently cloned a cDNA for glycogen synthase (gsn) from Neurospora crassa, and showed that gsn transcription decreased when cells were exposed to heat shock (shifted from 30degreesC to 45degreesC). In order to understand the mechanisms that control gsn expression, we isolated the gene, including its 5' and 3' flanking regions, from the genome of N. crassa. An ORF of approximately 2.4 kb was identified, which is interrupted by four small introns (II-V). Intron I (482 bp) is located in the 5'UTR region. Three putative Transcription Initiation Sites (TISs) were mapped, one of which lies downstream of a canonical TATA-box sequence (5'-TGTATAAA-3'). Analysis of the 5'-flanking region revealed the presence of putative transcription factor-binding sites, including Heat Shock Elements (HSEs) and STress Responsive Elements (STREs). The possible involvement of these motifs in the negative regulation of gsn transcription was investigated using Electrophoretic Mobility Shift Assays (EMSA) with nuclear extracts of N. crassa mycelium obtained before and after heat shock, and DNA fragments encompassing HSE and STRE elements from the 5'-flanking region. While elements within the promoter region are involved in transcription under heat shock, elements in the 5'UTR intron may participate in transcription during vegetative growth. The results thus suggest that N. crassa possesses trans-acting elements that interact with the 5'-flanking region to regulate gsn transcription during heat shock and vegetative growth.