Nerve-Derived Trophic Factors and DNA Elements Controlling Expression of Genes Encoding Synaptic Proteins in Skeletal Muscle Fibers

The neuromuscular junction represents an excellent model system for studying various critical issues in neurobiology at the molecular, cellular, and physiological levels. Our understanding of the basic events underlying synpase formation, maintenance, and plasticity has progressed considerably over the last few years primarily because of the numerous studies that have focused on this synapse and used sophisticated recombinant DNA technology. Recent data indicate that myonuclei located in the vicinity of the postsynaptic membrane are in a differential state of transcription compared to nuclei of the extrasynaptic sarcoplasm. Thus, renewal of postsynaptic membrane proteins appears to occur via a mechanism involving the local transcriptional activation of genes encoding these specialized proteins and extracellular cues originating from motoneurons. Such interaction between presynaptic nerve terminals and the postsynaptic sarcoplasm indicates that the entire signal transduction pathway is compartmentalized at the level of the neuromuscular junction. Expression of these genes appears less coregulated than originally anticipated, indicating that maintenance of the postsynaptic membrane requires the contribution of multiple extracellular signals, which ultimately urge target transcription factors to distinct DNA regulatory elements via various second messenger systems. Key words: neuromuscular junction, synapse, acetylcholinesterase, utrophin, agrin, CGRP, promoter, mRNA

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Characterization of the C-terminal extension of carboxysomal carbonic anhydrase from Synechocystis sp. PCC6803

Functional Plant Biology ◽

10.1071/pp01179 ◽

2002 ◽

Vol 29 (3) ◽

pp. 183 ◽

Cited By ~ 21

Author(s):

Anthony K.-C. So ◽

Swan S.-W. Cot ◽

George S. Espie

Keyword(s):

Carbonic Anhydrase ◽

Protein Interaction ◽

Recombinant Dna ◽

Carbonic Anhydrases ◽

Recombinant Dna Technology ◽

Higher Plant ◽

High Sequence Identity ◽

Synechocystis Pcc6803 ◽

Plant Enzymes ◽

Genes Encoding

Sequence analysis of the carboxysomal carbonic anhydrase (CcaA) from Synechocystis PCC6803, Synechococcus PCC7942 and Nostoc ATCC29133, indicated high sequence identity to the β class of plant and bacterial carbonic anhydrases (CA), and conservation of the active site region. However, the cyanobacterial enzyme has a C-terminal extension of about 75 amino acids (aa) not found in the plant enzymes, and largely absent from other bacterial enzymes. Using recombinant DNA technology, genes encoding C-terminal truncation products of up to 127 aa were overexpressed in E. coli, and partially purified lysates were analysed for CA-mediated exchange of 18O between 13C18O2and H216O. Recombinant CcaA proteins with up to 60 aa removed (CcaAΔ60) were catalytically competent, but beyond this there was an abrupt loss of activity. CcaAΔ0, along with CcaAΔ40 and CcaAΔ60, also catalysed the hydrolysis of carbon oxysulfide (COS; an isoelectronic structural analogue of CO2), but CcaAΔ63 and CcaAΔ127 did not, indicating that truncations greater than 62 aa resulted in a general loss of catalytic competency. Analysis of protein-protein interaction using the yeast two-hybrid system revealed that CcaA did not interact with the large or small Rubisco subunits (RbcL and RbcS, respectively) of Synechocystis, but there was strong CcaA-CcaA interaction. This protein interaction also ceased with C-terminal truncations in CcaA greater than 60 aa. The correlation between loss of CcaA-CcaA interaction and CcaA catalytic activity suggests that the proximal portion of the C-terminal extension is required for oligomerization, and that this oligomerization is essential for catalysis by the cyanobacterial enzyme. Thus, the C-terminal extension may play an important role in the function of CA within cyanobacterial carboxysomes, which is not required by the higher plant enzymes.

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Phytochrome signal-transduction: characterization of pathways and isolation of mutants

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.1995.0139 ◽

1995 ◽

Vol 350 (1331) ◽

pp. 67-74 ◽

Cited By ~ 13

Keyword(s):

Gene Expression ◽

Signal Transduction ◽

Red Light ◽

Signal Transduction Pathway ◽

Regulatory Elements ◽

Signal Transduction Pathways ◽

Expression Of Genes ◽

Genes Encoding ◽

Regulated Gene Expression ◽

Dependent Pathway

The study of phytochrome signalling has yielded a wealth of data describing both the perception of light by the receptor, and the terminal steps in phytochrome-regulated gene expression by a number of transcription factors. We are now focusing on establishing the intervening steps linking phytochrome photoactivation to gene expression, and the regulation and interactions of these signalling pathways. Recent work has utilized both a pharmacological approach in phototrophic soybean suspension cultures and microinjection techniques in tomato to establish three distinct phytochrome signal-transduction pathways: (i) a calcium-dependent pathway that regulates the expression of genes encoding the chlorophyll a/b binding protein ( CAB ) and other components of photosystem II; (ii) a cGMP-dependent pathway that regulates the expression of the gene encoding chalcone synthase ( CHS ) and the production of anthocyanin pigments; and (iii) a pathway dependent upon both calcium and cGMP that regulates the expression of genes encoding components of photosystem I and is necessary for the production of mature chloroplasts. To study the components and the regulation of phytochrome signal-transduction pathways, mutants with altered photomorphogenic responses have been isolated by a number of laboratories. However, with several possible exceptions, little real progress has been made towards the isolation of mutants in positive regulatory elements of the phytochrome signal-transduction pathway. We have characterized a novel phytochrome A (phyA)-mediated far-red light (FR) response in Arabidopsis seedlings which we are currently using to screen for specific phyA signal-transduction mutants.

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Core Element Cloning, Cis-Element Mapping and Serum Regulation of the Human EphB4 Promoter: A Novel TATA-Less Inr/MTE/DPE-Like Regulated Gene

Genes ◽

10.3390/genes10120997 ◽

2019 ◽

Vol 10 (12) ◽

pp. 997 ◽

Cited By ~ 1

Author(s):

Pierluigi Scalia ◽

Stephen J. Williams ◽

Antonio Giordano

Keyword(s):

Recombinant Dna ◽

Core Promoter ◽

Gene Promoter ◽

Bioinformatic Analysis ◽

Regulatory Elements ◽

Upstream Region ◽

Tyrosine Kinase Receptor ◽

Recombinant Dna Technology ◽

Core Element ◽

Cis Acting

The EphB4 gene encodes for a transmembrane tyrosine kinase receptor involved in embryonic blood vessel differentiation and cancer development. Although EphB4 is known to be regulated at the post-translational level, little is known about its gene regulation. The present study describes the core promoter elements’ identification and cloning, the cis-regulatory elements’ mapping and the serum regulation of the human EphB4 gene promoter region. Using bioinformatic analysis, Sanger sequencing and recombinant DNA technology, we analyzed the EphB4 gene upstream region spanning +40/−1509 from the actual transcription start site (TSS) and proved it to be a TATA-less gene promoter with dispersed regulatory elements characterized by a novel motif-of-ten element (MTE) at positions +18/+28, and a DPE-like motif and a DPE-like-repeated motif (DRM) spanning nt +27/+30 and +32 +35, respectively. We also mapped both proximal (multiple Sp1) and distal (HoxA9) trans-activating/dispersed cis-acting transcription factor (TF)-binding elements on the region we studied and used a transient transfection reporter assay to characterize its regulation by serum and IGF-II using EphB4 promoter deletion constructs with or without the identified new DNA-binding elements. Altogether, these findings shed new light on the human EphB4 promoter structure and regulation, suggesting mechanistic features conserved among Pol-II TATA-less genes phylogenetically shared from Drosophila to Human genomes.

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p67 isoform of mouse disabled 2 protein acts as a transcriptional activator during the differentiation of F9 cells

Biochemical Journal ◽

10.1042/bj3520645 ◽

2000 ◽

Vol 352 (3) ◽

pp. 645-650 ◽

Cited By ~ 15

Author(s):

Si Young CHO ◽

Jae Won JEON ◽

Sang Ho LEE ◽

Sung Soo PARK

Keyword(s):

Transcriptional Activation ◽

Biological Function ◽

Signal Transduction Pathway ◽

Activation Function ◽

Regulatory Elements ◽

Cell Nuclei ◽

Induced Differentiation ◽

Interacting Protein ◽

F9 Cells ◽

Transcriptional Regulatory Elements

The mouse disabled 2 (mDab2) gene is a mouse homologue of the Drosophila disabled gene and is alternatively spliced to form two isoforms, p96 and p67. Although p96 has been known to regulate the RasŐSos G-protein signal transduction pathway by interacting with Grb2, little is known about the biological function of p67. Recent studies have shown that the expression of mDab2 is markedly up-regulated during the retinoic acid (RA)-induced differentiation of F9 cells, suggesting another role for mDab2 in cell differentiation [Cho, Lee and Park (1999) Mol. Cells 9, 179Ő184). In the present study, we first elucidated the biological function of p67 isoform of mDab2 and identified its binding partner. Unlike p96, p67 largely resides in RA-treated F9 cell nuclei. In this system, p67 interacts with mouse androgen-receptor interacting protein 3, termed the mDab2 interacting protein, which acts as a transcriptional co-regulator. By using a fusion protein with a heterologous DNA-binding domain (GAL4), we showed that p67 had an intrinsic transcriptional activation function. These results suggest that mDab2 p67 may function as a transcriptional co-factor for certain complexes of transcriptional regulatory elements involved in the RA-induced differentiation of F9 cells.

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Neural regulation of acetylcholinesterase mRNAs at mammalian neuromuscular synapses.

The Journal of Cell Biology ◽

10.1083/jcb.127.4.1061 ◽

1994 ◽

Vol 127 (4) ◽

pp. 1061-1069 ◽

Cited By ~ 67

Author(s):

R N Michel ◽

C Q Vu ◽

W Tetzlaff ◽

B J Jasmin

Keyword(s):

Muscle Fibers ◽

Postsynaptic Membrane ◽

Synaptic Proteins ◽

Trophic Factors ◽

Mammalian Skeletal Muscle ◽

Mrna Levels ◽

Adult Muscle ◽

Neuromuscular Synapses ◽

Evoked Activity

We examined the role of innervation on acetylcholinesterase (AChE) gene expression within mammalian skeletal muscle fibers. First, we showed the selective accumulation of AChE mRNAs within the junctional vs extrajunctional sarcoplasm of adult muscle fibers using a quantitative reverse transcription PCR assay and demonstrated by in situ hybridization experiments that AChE transcripts are concentrated immediately beneath the postsynaptic membrane of the neuromuscular junction. Next, we determined the influence of nerve-evoked activity vs putative trophic factors on the synaptic accumulation of AChE mRNA levels in muscle fibers paralyzed by either surgical denervation or selective blockage of nerve action potentials with chronic superfusion of tetrodotoxin. Our results indicated that muscle paralysis leads to a marked decrease in AChE transcripts from the postsynaptic sarcoplasm, yet the extent of this decrease is less pronounced after tetrodotoxin inactivation than after denervation. These results suggest that although nerve-evoked activity per se appears a key regulator of AChE mRNA levels, the integrity of the synaptic structure or the release of putative trophic factors contribute to maintaining the synaptic accumulation of AChE transcripts at adult neuromuscular synapses. Furthermore, the pronounced downregulation of AChE transcripts in paralyzed muscles stands in sharp contrast to the well-documented increase in nicotinic acetylcholine receptor mRNAs under these conditions, and indicates that expression of the genes encoding these two synaptic proteins are subjected to different regulatory mechanisms in adult muscle fibers in vivo.

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The engulfment receptor Draper organizes the postsynaptic spectrin cytoskeleton into corrals containing synaptic proteins and promotes synaptic renewal

10.1101/846121 ◽

2019 ◽

Author(s):

Simon Wang ◽

Mannan Wang ◽

Hae-yoon Kim ◽

Nicole Yoo ◽

Matias Raski ◽

...

Keyword(s):

Neuromuscular Junction ◽

Motor Neurons ◽

Postsynaptic Membrane ◽

Synaptic Proteins ◽

Muscle Membrane ◽

Lateral Mobility ◽

Present Evidence ◽

Spectrin Cytoskeleton ◽

Symmetric Patterns

SummaryThe spectrin cytoskeleton is required for development of the Drosophila neuromuscular junction (NMJ) but its role is unclear. Here we show that the muscle spectrin lattice functions to corral membrane-associated synaptic proteins and limit their lateral mobility. Drosophila adducin, Hts, is required for integrity of the spectrin cytoskeleton and disruption of Hts function results in failure of the corrals. The spectrin cytoskeleton is itself patterned at the muscle membrane by the engulfment receptor Draper (Drpr) through regulation of Hts. We find patches of membrane where the spectrin cytoskeleton is organized into bilaterally symmetric patterns, which coincide with a field of Drpr-dependent structures similar to phagocytic pseudopods. The bilaterally symmetric patterns are likely created by folds of the muscle membrane in the pseudopods. We present evidence that the folds trap nascent boutons of motor neurons, leading to boutons with a bilaterally symmetric organization of the postsynaptic membrane. Drpr thus acts as a sensor of synaptic damage that promotes synaptogenesis.

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Isolation, Partial Purification and Characterization of a Novel Restriction Enzyme from Pseudomonas Anguilliseptica

10.21203/rs.3.rs-600889/v1 ◽

2021 ◽

Author(s):

Swarna Nirosha Jayasinghe Pathirana ◽

Don Anushka Sandaruwan Elvitigala ◽

Chandrika Malkanthi Nanayakkara ◽

Prashanth Suravajhala ◽

Sanath Rajapakse ◽

...

Keyword(s):

16S Rrna ◽

Recombinant Dna ◽

Bacterial Species ◽

Evolutionary Relationship ◽

Restriction Enzymes ◽

Partial Purification ◽

Recombinant Dna Technology ◽

Recognition Sequence ◽

16S Rrna Analysis ◽

Genes Encoding

Abstract Type II restriction enzymes (REs) which can cleave double stranded DNA in a sequence specific manner have many applications in recombinant DNA technology and are considered the work horses of molecular biology. Soil and water samples were screened for isolation of bacteria, harboring restriction enzymes. Cell lysates of isolated bacteria were incubated with unmethylated λ DNA, followed by analysis by agarose gel electrophoresis. The presence of distinct banding patterns indicated the presence of REs. Nine putative isolates harboring REs were morphologically and molecularly characterized using 16S rRNA analysis and belonged to four different genera (Acinetobacter, Lysinibacillus, Pseudomonas, and Brevibacillus). A HindIII like restriction digestion profile was observed in a lysate of a soil bacterium belonging to genus Pseudomonas. Based on 16S rRNA analysis, the bacterial species was identified as P. angulliseptica. The enzyme was partially purified and optimum conditions for enzyme activity and its recognition sequence were determined. The enzyme showed optimum activity at 40 0C and was stable at 40 °C for 20 minutes without the DNA substrate. The Recognition sequence of the enzyme was determined and found to be 5’AAGCT 3’ indicating it to be an isoschizomer of HindIII. The whole genome of the Pseudomonas species was sequenced and the coding sequence of the gene for the putative HindIII isoschizomer was identified together with other genes encoding putative REs. The gene coding for the HindIII isoschizomer was analyzed in silico and its homology and evolutionary relationship to other known isoschizomers of HindIII were determined. The enzyme was tentatively designated as PanI.

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Medicinal Plants: Guests and Hosts in the Heterologous Expression of High-Value Products

Planta Medica ◽

10.1055/a-1576-4148 ◽

2021 ◽

Author(s):

Nikolay Vasilev

Keyword(s):

Medicinal Plants ◽

Heterologous Expression ◽

Recombinant Dna ◽

Recombinant Protein Expression ◽

Regulatory Elements ◽

Pharmaceutical Products ◽

Scale Production ◽

Plant Metabolites ◽

Recombinant Dna Technology ◽

Success Stories

AbstractMedicinal plants play an important dual role in the context of the heterologous expression of high-value pharmaceutical products. On the one hand, the classical biochemical and modern omics approaches allowed for the discovery of various genes encoding biosynthetic pathways in medicinal plants. Recombinant DNA technology enabled introducing these genes and regulatory elements into host organisms and enhancing the heterologous production of the corresponding secondary metabolites. On the other hand, the transient expression of foreign DNA in plants facilitated the production of numerous proteins of pharmaceutical importance. This review summarizes several success stories of the engineering of plant metabolic pathways in heterologous hosts. Likewise, a few examples of recombinant protein expression in plants for therapeutic purposes are also highlighted. Therefore, the importance of medicinal plants has grown immensely as sources for valuable products of low and high molecular weight. The next step ahead for bioengineering is to achieve more success stories of industrial-scale production of secondary plant metabolites in microbial systems and to fully exploit plant cell factoriesʼ commercial potential for recombinant proteins.

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Regulation of Acetylcholine Receptor Biosynthesis During Motor Endplate Morphogenesis

Physiology ◽

10.1152/physiologyonline.1989.4.1.5 ◽

1989 ◽

Vol 4 (1) ◽

pp. 5-9

Author(s):

R Laufer ◽

B Fontaine ◽

A Klarsfeld ◽

J Cartaud ◽

J-P Changeux

Keyword(s):

Acetylcholine Receptor ◽

Synapse Formation ◽

Recombinant Dna ◽

Motor Endplate ◽

Synaptic Proteins ◽

Recombinant Dna Technology ◽

Dna Technology

Tools from recombinant DNA technology are now available to analyse the mechanisms that regulate the expression of synaptic proteins during synapse formation and stabilization. This review focuses on the expression of the acetylcholine receptor, an essential and well-characterized component of the motor endplate.

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Enhancing the Sweetness of Yoghurt through Metabolic Remodeling of Carbohydrate Metabolism in Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus

Applied and Environmental Microbiology ◽

10.1128/aem.00462-16 ◽

2016 ◽

Vol 82 (12) ◽

pp. 3683-3692 ◽

Cited By ~ 17

Author(s):

Kim I. Sørensen ◽

Mirjana Curic-Bawden ◽

Mette P. Junge ◽

Thomas Janzen ◽

Eric Johansen

Keyword(s):

Lactic Acid ◽

Recombinant Dna ◽

Streptococcus Thermophilus ◽

Lactobacillus Delbrueckii ◽

Phosphotransferase System ◽

Recombinant Dna Technology ◽

Kinase Gene ◽

Content Type ◽

Metabolic Remodeling ◽

Genes Encoding

ABSTRACTStreptococcus thermophilusandLactobacillus delbrueckiisubsp.bulgaricusare used in the fermentation of milk to produce yoghurt. These species normally metabolize only the glucose moiety of lactose, secreting galactose and producing lactic acid as the main metabolic end product. We used multiple serial selection steps to isolate spontaneous mutants of industrial strains ofS. thermophilusandL. delbrueckiisubsp.bulgaricusthat secreted glucose rather than galactose when utilizing lactose as a carbon source. Sequencing revealed that theS. thermophilusstrains had mutations in thegalKTEMpromoter, the glucokinase gene, and genes encoding elements of the glucose/mannose phosphotransferase system (PTS). These strains metabolize galactose but are unable to phosphorylate glucose internally or via the PTS. TheL. delbrueckiisubsp.bulgaricusmutants had mutations in genes of the glucose/mannose PTS and in the pyruvate kinase gene. These strains cannot grow on exogenous glucose but are proficient at metabolizing internal glucose released from lactose by β-galactosidase. The resulting strains can be combined to ferment milk, producing yoghurt with no detectable lactose, moderate levels of galactose, and high levels of glucose. Since glucose tastes considerably sweeter than either lactose or galactose, the sweetness of the yoghurt is perceptibly enhanced. These strains were produced without the use of recombinant DNA technology and can be used for the industrial production of yoghurt with enhanced intrinsic sweetness and low residual levels of lactose.IMPORTANCEBased on a good understanding of the physiology of the lactic acid bacteriaStreptococcus thermophilusandLactobacillus delbrueckiisubsp.bulgaricus, we were able, by selecting spontaneously occurring mutants, to change dramatically the metabolic products secreted into the growth medium. These mutants consume substantially more of the lactose, metabolize some of the galactose, and secrete the remaining galactose and most of the glucose back into the milk. This allows production of yoghurt with very low lactose levels and enhanced natural sweetness, because humans perceive glucose as sweeter than either lactose or galactose.

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