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Macrolide and Tetracycline Resistance and Molecular Relationships of Clinical Strains of Streptococcus agalactiae

Culebras, Esther; Rodriguez-Avial, Iciar; Betriu, Carmen; Redondo, Montserrat; Picazo, Juan J.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /05/2002 Português
Relevância na Pesquisa
140.000625%
Mechanisms for tetracycline and macrolide resistance in 54 isolates of erythromycin-resistant Streptococcus agalactiae were analyzed by PCR. The erm(B), erm(A), and mef(A) genes, either alone or in combination, were detected in all the erythromycin-resistant isolates. The tet(M) and tet(O) genes were responsible for tetracycline resistance. Random amplification of polymorphic DNA indicated different clonal origins of the isolates.

Resistance Determinants and Clonal Diversity in Group A Streptococci Collected during a Period of Increasing Macrolide Resistance

Cresti, Stefania; Lattanzi, Maria; Zanchi, Alessandra; Montagnani, Francesca; Pollini, Simona; Cellesi, Carla; Rossolini, Gian Maria
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /06/2002 Português
Relevância na Pesquisa
140.000625%
Susceptibility to macrolides and lincosamides was investigated with 299 consecutive nonduplicate Streptococcus pyogenes clinical isolates collected over a 6-year period (1992 to 1997) from an area of central Italy. During this period, macrolide resistance rates steadily increased (from 9% in 1992 to 53% in 1997; P < 0.001). The increase was caused by isolates with a macrolide-lincosamide-streptogramin B resistance phenotype, carrying mostly erm(B) but also erm(TR) genes, that were not detected in the first 2 years and were detected with increasing prevalence (8, 5, 26, and 37%, respectively) during the following 4 years. During the same period, the prevalence of isolates with a macrolide resistance phenotype, carrying mef(A) determinants, did not vary significantly; on average it was 13%, with modest rate fluctuations in different years and no definite trend. Molecular typing revealed a remarkable clonal diversity among susceptible and resistant isolates and a notable heterogeneity of the genetic environment of the resistance genes. The analysis of clonal diversity in relation with resistance phenotypes and genotypes revealed that increased macrolide resistance rates were due to a complex interplay of different mechanisms, with a relevant contribution played by an “epidemic” spread of genetic elements carrying the erm(B) gene among the circulating streptococcal population.

Topology of the Ca2+ release channel of skeletal muscle sarcoplasmic reticulum (RyR1)

Du, Guo Guang; Sandhu, Bimal; Khanna, Vijay K.; Guo, Xing Hua; MacLennan, David H.
Fonte: National Academy of Sciences Publicador: National Academy of Sciences
Tipo: Artigo de Revista Científica
Português
Relevância na Pesquisa
140.000625%
To define the topology of the skeletal muscle ryanodine receptor (RyR1), enhanced GFP (EGFP) was fused in-frame to the C terminus of RyR1, replacing a series of C-terminal deletions that started near the beginning or the end of predicted transmembrane helices M1–M10. The constructs were expressed in HEK-293 (human embryonic kidney cell line 293) or mouse embryonic fibroblast (MEF) cells, and confocal microscopy of intact and saponin-permeabilized cells was used to determine the subcellular location of the truncated fusion proteins. The fusion protein truncated after M3 exhibited uniform cytoplasmic fluorescence, which was lost after permeabilization, indicating that proposed M′, M′′, M1, M2, and M3 sequences are not membrane-associated. The fusion protein truncated at the end of the M4–M5 loop and containing M4 was membrane-associated. All longer truncated fusion proteins were also associated with intracellular membranes. Mapping by protease digestion and extraction of isolated microsomes demonstrated that EGFP positioned after either M5, the N-terminal half of M7 (M7a), or M8 was located in the lumen, and that EGFP positioned after either M4, M6, the C-terminal half of M7 (M7b), or M10 was located in the cytoplasm. These results indicate that RyR1 contains eight transmembrane helices...

High Rate of Transmission of Penicillin-Resistant Streptococcus pneumoniae between Parents and Children

Hoshino, Kazuhiko; Watanabe, Hiroshi; Sugita, Rinya; Asoh, Norichika; Ntabaguzi, Simon Angelo; Watanabe, Kiwao; Oishi, Kazunori; Nagatake, Tsuyoshi
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /11/2002 Português
Relevância na Pesquisa
140.000625%
Transmission of Streptococcus pneumoniae between children and their parents was evaluated in 29 pairs from 25 families. The serotypes of 35 pneumococcal isolates from 18 (62.1%) of 29 child-parent pairs were identical. Of the 35 isolates, 23 showed intermediate resistance and 10 were fully resistant to penicillin G. PCR indicated that all 35 strains had at least one alteration in penicillin-binding protein genes pbp1a, pbp2x, and pbp2b and 33 strains had macrolide resistance genes mef(A) and/or erm(B). As a result, the PCR patterns of 16 of 18 pairs were identical. Molecular typing by pulsed-field gel electrophoresis showed that 12 pairs were indistinguishable, 3 pairs were closely related, 2 pairs were possibly related, and only one pair was different. Our data indicate the presence of a high rate of transmission of penicillin-resistant S. pneumoniae between children and their parents.

Macrolide-Resistant Streptococcus pneumoniae and Streptococcus pyogenes in the Pediatric Population in Germany during 2000-2001

Reinert, Ralf René; Lütticken, Rudolf; Bryskier, André; Al-Lahham, Adnan
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /02/2003 Português
Relevância na Pesquisa
140.000625%
In a nationwide study in Germany covering 13 clinical microbiology laboratories, a total of 307 Streptococcus pyogenes (mainly pharyngitis) and 333 Streptococcus pneumoniae (respiratory tract infections) strains were collected from outpatients less than 16 years of age. The MICs of penicillin G, amoxicillin, cefotaxime, erythromycin A, clindamycin, levofloxacin, and telithromycin were determined by the microdilution method. In S. pyogenes isolates, resistance rates were as follows: penicillin, 0%; erythromycin A, 13.7%; and levofloxacin, 0%. Telithromycin showed good activity against S. pyogenes isolates (MIC90 = 0.25 μg/ml; MIC range, 0.016 to 16 μg/ml). Three strains were found to be telithromycin-resistant (MIC ≥ 4 μg/ml). Erythromycin-resistant strains were characterized for the underlying resistance genotype, with 40.5% having the efflux type mef(A), 38.1% having the erm(A), and 9.5% having the erm(B) genotypes. emm typing of macrolide-resistant S. pyogenes isolates showed emm types 4 (45.2%), 77 (26.2%), and 12 (11.9%) to be predominant. In S. pneumoniae, resistance rates were as follows: penicillin intermediate, 7.5%; penicillin resistant, 0%; erythromycin A, 17.4%; and levofloxacin, 0%. Telithromycin was highly active against pneumococcal isolates (MIC90 ≤ 0.016 μg/ml; range...

Genetic Basis of Antibiotic Resistance in Streptococcus agalactiae Strains Isolated in a French Hospital

Poyart, Claire; Jardy, Laurence; Quesne, Gilles; Berche, Patrick; Trieu-Cuot, Patrick
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /02/2003 Português
Relevância na Pesquisa
140.000625%
The genetic basis of antibiotic resistance in 113 unrelated group B streptococci was studied by PCR. Ninety-four strains were resistant to tetracycline-minocycline, and tet(M) was detected in 85% of these isolates. Seventeen erythromycin-resistant strains contained the erm(B), erm(TR), or mef(A) gene. Eleven strains exhibited high-level resistance to kanamycin due to the presence of the aphA3 gene; eight of these strains were also highly resistant to streptomycin; aad-6-related sequences were detected in seven strains.

Antimicrobial Resistance of Invasive Pneumococci in Finland in 1999-2000

Pihlajamäki, Marja; Jalava, Jari; Huovinen, Pentti; Kotilainen, Pirkko;
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /06/2003 Português
Relevância na Pesquisa
140.000625%
The resistance patterns and macrolide resistance mechanisms of 910 Finnish invasive pneumococci isolated during 1999 and 2000 were studied. Macrolide resistance was detected in 6.9% of isolates. Penicillin resistance was detected in 1.5% of isolates, and penicillin intermediate resistance was detected in 4.0% of isolates. Active macrolide efflux, mediated by the mef(A) gene, was the most common macrolide resistance mechanism. Four macrolide-resistant isolates had mutations in rRNA or ribosomal protein L22.

Specific protein redirection as a transcriptional therapy approach for t(8;21) leukemia

Steffen, Björn; Serve, Hubert; Berdel, Wolfgang E.; Agrawal, Shuchi; Linggi, Bryan; Büchner, Thomas; Hiebert, Scott W.; Müller-Tidow, Carsten
Fonte: National Academy of Sciences Publicador: National Academy of Sciences
Tipo: Artigo de Revista Científica
Português
Relevância na Pesquisa
140.000625%
Important progress has been achieved in the knowledge about the pathogenesis of cancer. However, despite these advances, the therapeutic strategies are still limited. Leukemias are often characterized by specific balanced translocations, with the t(8;21) balanced translocation being the most frequent chromosomal aberration in acute myeloid leukemia (AML). This translocation produces the AML1-ETO fusion protein, which binds to AML1 target promoter sequences. Transcriptional repression of AML1-dependent genes by AML1-ETO and associated corepressors represents the pathogenetic mechanisms of t(8;21). Here, we show that targeting of AML1-ETO to essential, MYB-dependent gene promoters induces t(8;21)-restricted cell death. We constructed a chimeric protein that contained the MYB DNA-binding domain and the AML1-binding domain of myeloid Elf-1-like factor (MEF). This protein associated with AML1-ETO and directed the complex to MYB-responsive promoters in vitro and in vivo. In the presence of AML1-ETO, the chimeric protein repressed the activity of MYB-responsive promoters, rapidly induced apoptosis, and specifically inhibited colony growth. All these effects occurred only in AML1-ETO-positive cells, whereas no adverse effects were observed in cells not expressing AML1-ETO. Taken together...

Oral efficacy of WIN 51711 in mice infected with human poliovirus.

McKinlay, M A; Steinberg, B A
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /01/1986 Português
Relevância na Pesquisa
140.000625%
WIN 51711, a new broad-spectrum anti-picornavirus agent, prevented the development of paralysis and subsequent death in mice infected intracerebrally with a lethal dose of human poliovirus type 2 (MEF strain). The prophylactic efficacy of intragastrically administered WIN 51711 was dose dependent over the 3.9- to 62.5-mg/kg (twice daily) dose range, with a minimal significantly effective dose of less than 15.6 mg/kg per dose (twice daily) (P less than 0.008). An oral four times a day dosage regimen initiated 48 h postinfection with WIN 51711 doses as low as 12.5 mg/kg was effective in significantly reducing poliovirus-induced paralysis and death compared with a placebo. Viral titers in the brains and spines of mice infected intracerebrally with 200 50% lethal doses of poliovirus were reduced by 3 to 5 log10 PFU/g in the WIN 51711-medicated group compared with placebo-medicated animals. The potent in vitro and in vivo anti-picornavirus activity of WIN 51711 makes it a potentially useful drug for the treatment of enterovirus infections in humans.

Prevention of human poliovirus-induced paralysis and death in mice by the novel antiviral agent arildone.

McKinlay, M A; Miralles, J V; Brisson, C J; Pancic, F
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /12/1982 Português
Relevância na Pesquisa
140.000625%
Arildone, a novel antiviral agent which blocks virion uncoating, was assessed for its ability to prevent paralysis and death in mice infected intracerebrally with a lethal dose of human poliovirus type-2 (strain MEF). Intraperitoneal administration of arildone suspended in gum tragacanth prevented paralysis and death in a dose-dependent manner (minimal inhibitory dose = 32 mg/kg, twice daily) and protected animals from virus challenges in excess of 20 50% lethal doses. Oral medication with arildone solubilized in corn oil was similarly effective in preventing poliovirus-induced paralysis and death. Arildone was therapeutically effective even when intraperitoneal medication was delayed for 48 h postinfection. Analysis of the virus titers in the central nervous system tissues of animals infected with 200 50% lethal doses demonstrated that arildone reduced titers in the brain and spine by approximately 3 and 4 log10 PFU per g of tissue, respectively, implying that direct inhibition of virus replication was responsible for host survival. Arildone is the first antiviral agent capable of preventing poliovirus-induced death in mice. The efficient inhibition of poliovirus replication described here demonstrates the potential usefulness of uncoating blockers in the systemic treatment of viral diseases.

Correlation between poliovirus type 1 Mahoney replication in blood cells and neurovirulence.

Freistadt, M S; Eberle, K E
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /09/1996 Português
Relevância na Pesquisa
140.000625%
Poliovirus (PV) is not often described as a monocyte- or macrophage-tropic virus; however, previous work indicated that neurovirulent PV type 1 Mahoney [PV(1)Mahoney] can productively infect primary human monocytes. To determine whether this replication has a functional role in pathogenesis, primary human mononuclear blood cells were infected with pairs of attenuated and neurovirulent strains of PV. Two neurovirulent strains of PV, PV(1)Mahoney and PV(2)MEF-1, replicated faster and to higher titers than attenuated counterparts PV(1)Sabin and PV(2)W-2, respectively, in primary human monocytes, suggesting that this replication may contribute to pathogenesis. PV(3)Leon grew weakly, while PV(3)Sabin, PV(2)Sabin, and PV(2) P712 did not replicate in these cells, perhaps because of their slow replication cycle. In U937 cells, a monocytelike cell line, PV(1)Mahoney replicated but PV(1)Sabin did not, while both grew well in HeLa cells. When molecular recombinants of PV(1)Mahoney and PV(1)Sabin were assessed, a correlation between neurovirulence and the ability to replicate in primary human mononuclear blood cells was found. Surprisingly, infectious centers assays with primary human mononuclear blood cells and U937 cells indicated that despite the lower overall viral yield...

Telomere-based crisis: functional differences between telomerase activation and ALT in tumor progression

Chang, Sandy; Khoo, Christine M.; Naylor, Maria L.; Maser, Richard S.; DePinho, Ronald A.
Fonte: Cold Spring Harbor Laboratory Press Publicador: Cold Spring Harbor Laboratory Press
Tipo: Artigo de Revista Científica
Publicado em 01/01/2003 Português
Relevância na Pesquisa
140.000625%
Telomerase activation is a common feature of most advanced human cancers and is postulated to restore genomic stability to a level permissive for cell viability and tumor progression. Here, we used genetically defined transformed mouse embryonic fibroblast (MEF) cultures derived from late generation mTerc−/− Ink4a/Arf−/− mice to explore more directly how telomere-based crisis relates to the evolution of cancer cell genomes and to tumor biology. An exhaustive serial analysis of cytogenetic profiles over extensive passage in culture revealed that the emergence of chromosomal fusions (including dicentrics) coincided with onset of deletions and complex nonreciprocal translocations (NRTs), whereas mTerc-transduced cultures maintained intact chromosomes and stable genomes. Despite a high degree of telomere dysfunction and genomic instability, transformed late passage mTerc−/− Ink4a/Arf−/− cultures retained the capacity to form subcutaneous tumors in immunocompromised mice. However, even moderate levels of telomere dysfunction completely abrogated the capacity of these cells to form lung metastases after tail-vein injection, whereas mTerc reconstitution alone conferred robust metastatic activity in these cells. Finally, serial subcutaneous tumor formation using late passage transformed mTerc−/− Ink4a/Arf−/− cultures revealed clear evidence of telomerase-independent alternative lengthening of telomeres (ALT). Significantly...

Common core sequences are found in skeletal muscle slow- and fast-fiber-type-specific regulatory elements.

Nakayama, M; Stauffer, J; Cheng, J; Banerjee-Basu, S; Wawrousek, E; Buonanno, A
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /05/1996 Português
Relevância na Pesquisa
140.000625%
The molecular mechanisms generating muscle diversity during development are unknown. The phenotypic properties of slow- and fast-twitch myofibers are determined by the selective transcription of genes coding for contractile proteins and metabolic enzymes in these muscles, properties that fail to develop in cultured muscle. Using transgenic mice, we have identified regulatory elements in the evolutionarily related troponin slow (TnIs) and fast (TnIf) genes that confer specific transcription in either slow or fast muscles. Analysis of serial deletions of the rat TnIs upstream region revealed that sequences between kb -0.95 and -0.5 are necessary to confer slow-fiber-specific transcription; the -0.5-kb fragment containing the basal promoter was inactive in five transgenic mouse lines tested. We identified a 128-bp regulatory element residing at kb -0.8 that, when linked to the -0.5-kb TnIs promoter, specifically confers transcription to slow-twitch muscles. To identify sequences directing fast-fiber-specific transcription, we generated transgenic mice harboring a construct containing the TnIs kb -0.5 promoter fused to a 144-bp enhancer derived from the quail TnIf gene. Mice harboring the TnIf/TnIs chimera construct expressed the transgene in fast but not in slow muscles...

Clinical and Molecular Epidemiology of Erythromycin-Resistant Beta-Hemolytic Lancefield Group G Streptococci Causing Bacteremia

Woo, Patrick C. Y.; To, Amanda P. C.; Tse, Herman; Lau, Susanna K. P.; Yuen, Kwok-yung
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /11/2003 Português
Relevância na Pesquisa
140.000625%
Among 100 patients with group G beta-hemolytic streptococcal bacteremia in a 6-year period (1997 to 2002), seven had bacteremia caused by erythromycin-resistant strains. Five of the seven patients had cellulitis and/or abscesses. The two isolates resistant to erythromycin and clindamycin possessed erm genes, one ermTR and the other ermB. The five isolates resistant to erythromycin but sensitive to clindamycin and one of those resistant to both erythromycin and clindamycin possessed mef genes.

In Vitro Activities of Tigecycline against Erythromycin-Resistant Streptococcus pyogenes and Streptococcus agalactiae: Mechanisms of Macrolide and Tetracycline Resistance

Betriu, C.; Culebras, E.; Rodríguez-Avial, I.; Gómez, M.; Sánchez, B. A.; Picazo, J. J.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /01/2004 Português
Relevância na Pesquisa
140.000625%
The activity of tigecycline was tested against erythromycin-resistant streptococci (107 Streptococcus pyogenes and 98 Streptococcus agalactiae strains). The presence of erythromycin and tetracycline resistance genes was determined by PCR. Among S. pyogenes strains the most prevalent gene was mef(A) (91.6%). The erm(B) gene was the most prevalent (65.3%) among S. agalactiae strains. Tigecycline proved to be very active against all the isolates tested (MIC at which 90% of the isolates tested were inhibited, 0.06 μg/ml), including those resistant to tetracycline.

IKK1-deficient mice exhibit abnormal development of skin and skeleton

Li, Qiutang; Lu, Qingxian; Hwang, Jason Y.; Büscher, Dirk; Lee, Kuo-Fen; Izpisua-Belmonte, Juan Carlos; Verma, Inder M.
Fonte: Cold Spring Harbor Laboratory Press Publicador: Cold Spring Harbor Laboratory Press
Tipo: Artigo de Revista Científica
Publicado em 15/05/1999 Português
Relevância na Pesquisa
140.000625%
IκB kinases (IKKs) IKK1 and IKK2 are two putative IκBα kinases involved in NF-κB activation. To examine the in vivo functions of IKK1, we generated IKK1-deficient mice. The mutant mice are perinatally lethal and exhibit a wide range of developmental defects. Newborn mutant mice have shiny, taut, and sticky skin without whiskers. Histological analysis shows thicker epidermis, which is unable to differentiate. Limbs and tail are wrapped inside the skin and do not extend properly out of the body trunk. Skeleton staining reveals a cleft secondary palate, split sternebra 6, and deformed incisors. NF-κB activation mediated by TNFα and IL-1 is diminished in IKK1-deficient mouse embryonic fibroblast (MEF) cells. The IKK complex in the absence of IKK1 is capable of phosphorylating IκBα and IκBβ in vitro. Our results support a role for IKK1 in NF-κB activation and uncover its involvement in skin and skeleton development. We conclude further that the two related kinases IKK1 and IKK2 have distinct functions and can not be substituted for each other’s functions.

Complete lack of NF-κB activity in IKK1 and IKK2 double-deficient mice: additional defect in neurulation

Li, Qiutang; Estepa, Gabriela; Memet, Sylvie; Israel, Alain; Verma, Inder M.
Fonte: Cold Spring Harbor Laboratory Press Publicador: Cold Spring Harbor Laboratory Press
Tipo: Artigo de Revista Científica
Publicado em 15/07/2000 Português
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140.000625%
NF-κB activity is induced by cytokines, stress, and pathogens. IKK1 and IKK2 are critical IκB kinases in NF-κB activation. In this study mice lacking IKK1 and IKK2 died at E12. Additional defect in neurulation associated with enhanced apoptosis in the neuroepithelium was also observed. MEF cells from IKK1−/−/IKK2−/− embryos did not respond to NF-κB inducers. Upon crossing with κB–lacZ transgenic mice, double-deficient embryos also lost lacZ transgene expression in vascular endothelial cells during development. Our data suggest that IKK1 and IKK2 are essential for NF-κB activation in vivo and have an important role in protecting neurons against excessive apoptosis during development.

Ovca1 regulates cell proliferation, embryonic development, and tumorigenesis

Chen, Chun-Ming; Behringer, Richard R.
Fonte: Cold Spring Harbor Laboratory Press Publicador: Cold Spring Harbor Laboratory Press
Tipo: Artigo de Revista Científica
Publicado em 01/02/2004 Português
Relevância na Pesquisa
140.000625%
Loss of OVCA1/DPH2L1 correlates with ovarian and breast cancer. To study its in vivo role, we generated Ovca1 mutant alleles in mice. Ovca1 heterozygotes spontaneously develop cancer. Ovca1 mutant mice die during embryonic development and at birth with developmental delay and defects in multiple organ systems. Cell proliferation defects were observed in Ovca1 mutant mouse embryonic fibroblasts (MEFs). p53 deficiency can rescue these Ovca1 mutant MEF proliferation defects and partially rescue Ovca1 mutant embryonic phenotypes. Furthermore, Ovca1; p53 double heterozygotes developed tumors quicker than p53 heterozygotes and with an increased carcinoma incidence. Multiple tumor burden in Ovca1 heterozygotes that were also p53 deficient was significantly higher than in p53 homozygous mutants. These in vivo findings demonstrate that Ovca1 is a tumor suppressor that can modify p53-induced tumorigenesis and suggest that it acts as a positive regulator for cell cycle progression. The close linkage of OVCA1 and p53 on human Chromosome 17 suggests that coordinated loss may be an important mechanism for the evolution of ovarian, breast, and other tumor phenotypes.

Positive regulatory elements (HF-1a and HF-1b) and a novel negative regulatory element (HF-3) mediate ventricular muscle-specific expression of myosin light-chain 2-luciferase fusion genes in transgenic mice.

Lee, K J; Hickey, R; Zhu, H; Chien, K R
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /02/1994 Português
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140.000625%
The cardiac myosin light-chain 2v (MLC-2v) gene has served as a model system to identify the pathways which restrict the expression of cardiac muscle genes to particular chambers of the heart during cardiogenesis. To identify the critical cis regulatory elements which mediate ventricular chamber-specific expression of the MLC-2v gene in the in vivo context, a series of transgenic mice which harbor mutations in putative MLC-2 cis regulatory elements in a 250-bp MLC-2-luciferase fusion gene which is expressed in a ventricular chamber-specific fashion in transgenic mice were generated. These studies demonstrate that both components of HF-1 (HF-1a and HF-1b/MEF-2) are required to maintain ventricular chamber-specific expression and function as positive regulatory elements. Mutations in another conserved element (HF-2) are without statistically significant effect on ventricular chamber expression. Transgenics harboring mutations in the E-box site also displayed significant upregulation of reporter activity in the soleus, gastrocnemius, and uterus, with a borderline effect on expression in liver. Mutations in another conserved element (HF-3) result in a marked (> 75-fold) upregulation of the luciferase reporter activity in the soleus muscle of multiple independent or transgenic founders. Since the HF-3 mutations appeared to have only a marginal effect on luciferase reporter activity in liver tissue...

A 40-kilodalton protein binds specifically to an upstream sequence element essential for muscle-specific transcription of the human myoglobin promoter.

Bassel-Duby, R; Hernandez, M D; Gonzalez, M A; Krueger, J K; Williams, R S
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /11/1992 Português
Relevância na Pesquisa
140.000625%
To define transcriptional control elements responsible for muscle-specific expression of the human myoglobin gene, we performed mutational analysis of upstream sequences (nucleotide positions -373 to +7 relative to the transcriptional start site) linked to a firefly luciferase gene. Transient expression assays in avian and mammalian cells indicated that a CCCACCCCC (CCAC box) sequence (-223 to -204) is necessary for muscle-specific transcription directed either by the native myoglobin promoter or by a heterologous minimal promoter linked to the myoglobin upstream enhancer region. A putative MEF2-like site (-160 to -169) was likewise necessary for full transcriptional activity in myotubes. Mutations within either of two CANNTG (E-box) motifs (-176 to -148) had only minimal effects on promoter function. We identified and partially purified from nuclear extracts a 40-kDa protein (CBF40) that binds specifically to oligonucleotides containing the CCAC box sequence. A mutation of the CCAC box that disrupted promoter function in vivo also impaired binding of CBF40 in vitro. These data suggest that cooperative interactions between CBF40 and other factors including MEF-2 are required for expression of the human myoglobin gene in skeletal muscle.