A novel cytoskeletal associated protein: Genomic organization and protein pattern

1988 ◽  
Vol 53 (1) ◽  
pp. 198-201 ◽  
Author(s):  
Haralabos Zorbas ◽  
Ernst Pöschl ◽  
Antonia Pöschl ◽  
Peter K. Müller
Keyword(s):  
Genomic Organization ◽  
Genomic Library ◽  
Protein Pattern ◽  
Genomic Structure ◽  
Initial Results

c-DNA clones were constructed from chicken sternal m-RNA and screened for recognizing those m-RNA species the amount of which decreases during chondrocyte dedifferentiation. One such clone was used to screen a chicken genomic library and, furthermore, to deduce peptide sequences suitable for raising antibodies. Initial results on the genomic structure and on some biochemical and immunochemical properties are presented.

Gene for integrin-associated protein (IAP, CD47): Physical mapping, genomic structure, and expression studies in skeletal muscle

2002 ◽  
Vol 80 (2) ◽  
pp. 169-176 ◽  
Author(s):  
Jörg Schickel ◽  
Katharina Stahn ◽  
Klaus-Peter Zimmer ◽  
Ralf Sudbrak ◽  
Tim M Størm ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Human Chromosome ◽  
Genomic Organization ◽  
Genomic Structure ◽  
Splice Form ◽  
Form Expression ◽  
Multiple Functions ◽  
Expression Studies ◽  
Tubular System ◽  
Neuronal Processes

Integrin-associated protein (IAP) is a widely expressed membrane protein with multiple functions in immunological and neuronal processes. Having physically mapped the IAP gene into a BAC/PAC contig covering approximately 1 Mb on human chromosome 3q13.1-q13.2, we determined the genomic organization of the gene, established its expression in skeletal muscle, and identified a novel splice variant. Our expression studies demonstrate expression of integrin-associated protein in the t-tubular system and the euchromatin of skeletal muscle cells where its function thus far is not known.Key words: integrin-associated protein, novel splice form, expression and subcellular localization in skeletal muscle, genomic organization.

scholarly journals Murine cystathionine γ-lyase: complete cDNA and genomic sequences, promoter activity, tissue distribution and developmental expression

2004 ◽  
Vol 381 (1) ◽  
pp. 113-123 ◽  
Author(s):  
Isao ISHII ◽  
Noriyuki AKAHOSHI ◽  
Xiao-Nian YU ◽  
Yuriko KOBAYASHI ◽  
Kazuhiko NAMEKATA ◽  
...  
Keyword(s):  
Tissue Distribution ◽  
Genomic Library ◽  
Polyclonal Antibodies ◽  
Core Promoter ◽  
Genomic Structure ◽  
Expression Patterns ◽  
Enzymic Activity ◽  
Developmental Expression ◽  
Structure Generation ◽  
Expression Levels

Cystathionine γ-lyase (CSE) is the last key enzyme in the trans-sulphuration pathway for biosynthesis of cysteine from methionine. Cysteine could be provided through diet; however, CSE has been shown to be important for the adequate supply of cysteine to synthesize glutathione, a major intracellular antioxidant. With a view to determining physiological roles of CSE in mice, we report the sequence of a complete mouse CSE cDNA along with its associated genomic structure, generation of specific polyclonal antibodies, and the tissue distribution and developmental expression patterns of CSE in mice. A 1.8 kb full-length cDNA containing an open reading frame of 1197 bp, which encodes a 43.6 kDa protein, was isolated from adult mouse kidney. A 35 kb mouse genomic fragment was obtained by λ genomic library screening. It contained promoter regions, 12 exons, ranging in size from 53 to 579 bp, spanning over 30 kb, and exon/intron boundaries that were conserved with rat and human CSE. The GC-rich core promoter contained canonical TATA and CAAT motifs, and several transcription factor-binding consensus sequences. The CSE transcript, protein and enzymic activity were detected in liver, kidney, and, at much lower levels, in small intestine and stomach of both rats and mice. In developing mouse liver and kidney, the expression levels of CSE protein and activity gradually increased with age until reaching their peak value at 3 weeks of age, following which the expression levels in liver remained constant, whereas those in kidney decreased significantly. Immunohistochemical analyses revealed predominant CSE expression in hepatocytes and kidney cortical tubuli. These results suggest important physiological roles for CSE in mice.

scholarly journals Molecular cloning of equine 17β-hydroxysteroid dehydrogenase type 1 and its downregulation during follicular luteinization in vivo

2007 ◽  
Vol 38 (1) ◽  
pp. 67-78 ◽  
Author(s):  
Kristy A Brown ◽  
Khampoune Sayasith ◽  
Nadine Bouchard ◽  
Jacques G Lussier ◽  
Jean Sirois
Keyword(s):  
Granulosa Cell ◽  
Genomic Library ◽  
Genomic Structure ◽  
Hydroxysteroid Dehydrogenase ◽  
Proximal Region ◽  
Proximal Promoter ◽  
Transcript Expression ◽  
Nuclear Extracts

The type 1 form of 17β-hydroxysteroid dehydrogenase (17βHSD1) was the first isoform to be identified and is capable of converting estrone to 17β-estradiol. This study was aimed at characterizing the molecular structure of the equine 17βHSD1 gene and cDNA, as well as its molecular regulation during human chorionic gonadotropin (hCG)-induced follicular luteinization/ovulation in vivo. The equine 17βHSD1 gene was cloned from an equine genomic library and shown to have a conserved genomic structure composed of six exons. Its cDNA sequence was also identified and coded for a 308 amino acid protein, 72.1–74.5% homologous to other mammalian orthologs. RT-PCR/Southern blot analyses were performed to study the regulation of the 17βHSD1 transcript in equine preovulatory follicles isolated between 0 and 39 h after hCG treatment. Results demonstrated the presence of high 17βHSD1 mRNA expression prior to hCG treatment with a marked decrease observed 12 h after hCG (P < 0.05). Analyses on isolated preparations of granulosa and theca interna cells identified the granulosa cell layer as the site of 17βHSD1 transcript expression and downregulation (P < 0.05). A 1412 bp fragment of the equine 17βHSD1 proximal promoter was sequenced and shown to contain many putative transcription factor binding sites. Electromobility shift assays (EMSA) using a fragment of the proximal promoter (−230/−30) and nuclear extracts prepared from granulosa cells isolated prior to hCG (0 h post-hCG) revealed the presence of a major complex, and results from competition assays suggest that steroidogenic factor-1 (SF-1), NFκB, GATA, and Sp1 cis-elements are involved. Supershift assays using an antibody against the p65 subunit of NFκB led to the displacement of the binding nuclear proteins to the DNA probe, whereas the use of an anti-equine SF-1 antibody demonstrated the clear formation of a DNA–protein–antibody complex, confirming the potential role of these transcription factors in EMSA results. Interestingly, a notable decrease in DNA binding was observed when granulosa cell nuclear extracts isolated 30 h post-hCG were used, which paralleled the decrease in 17βHSD1 transcript after hCG treatment. Thus, this study is the first to report the gonadotropin-dependent downregulation of 17βHSD1 transcript expression in a monoovulatory species, the presence and regulation of protein/DNA interactions in the proximal region of the 17βHSD1 promoter during gonadotropin treatment, and the characterization of the primary structure of equine 17βHSD1 cDNA and gene.

scholarly journals Genomic Organization and Evolution of Alternative Exons in a Drosophila Calcium Channel Gene

Genetics ◽  
1997 ◽  
Vol 145 (4) ◽  
pp. 1003-1013 ◽  
Author(s):  
Alexandre A Peixoto ◽  
Lee A Smith ◽  
Jeffrey C Hall
Keyword(s):  
Calcium Channel ◽  
Genomic Organization ◽  
Genomic Sequence ◽  
Genomic Structure ◽  
Voltage Gated ◽  
Channel Gene ◽  
Rates Of Evolution ◽  
Gene Coding ◽  
Mammalian Counterpart ◽  
Α1 Subunit

The genomic organization of a gene coding for an α1 subunit of a voltage-gated calcium channel of Drosophila melanogaster (Dmca1A) was determined. Thirty-four exons, distributed over 45 kb of genomic sequence, have been identified and mapped, including exons in three regions involved in alternative splicing and new sites potentially involved in RNA editing. The comparison of the intron/exon boundaries of this channel with a mammalian counterpart shows that the genomic structure of these two genes has remained fairly similar during evolution, with more than half of the Drosophila intron positions being perfectly conserved compared to the human channel. Phylogenetic analysis of the mutually exclusive alternative exons revealed that they have diverged considerably. It is suggested that this divergence, rather than reflecting evolutionary age, is the likely result of accelerated rates of evolution following duplication.

scholarly journals Towards the Creation of a Transgenic Possum Parasite

2021 ◽  
Author(s):  
◽  
Jan Newton-Howes
Keyword(s):  
Genomic Library ◽  
Protein Pattern ◽  
Brushtail Possum ◽  
Native Protein ◽  
Putative Promoter Region ◽  
Reading Frame ◽  
Native Flora ◽  
C Elegans ◽  
Infective Larvae ◽  
Initiator Codon

<p>The brushtail possum. Trichosurus vulpecula, is New Zealand's most serious vertebrate pest; possums destroy native flora and fauna and are vectors of bovine Tb. Conventional control is considered to be unsustainable and, in the long term, biological control is seen as the only solution to reducing possum numbers. The aim of this project is to contribute to the development of a self-disseminating vector that will spread a control molecule throughout the possum population reducing fecundity or increasing mortality. The possum-specific parasite Parastrongyloides trichosuri has considerable potential a-s such a vector. A protein from P. trichosuri specifically, was found to be antigenic in possums. The antibodies to this protein were purified from positive possum serum and used to detect the antigen on the surface of infective larvae but not in the excretory/secretory products of either larvae or adults. The protein was isolated from crude infective larvae and found to show homology to the heat-shock 70 family of proteins. Genomic DNA was extracted, an oligonucleotide probe made and a genomic library screened for the Hsp70 gene. Several positive clones were found and DNA isolated and sequenced from one such clone. Five kilo bases of unambiguous sequence was obtained in which was an open reading frame of 2 kb. Theoretical translation of this gave a protein of 64 amino acids with 80% homology to the Hsp70A protein of C. elegans. The region upstream of the ATG initiator codon was amplified and 1.3 kb of the putative promoter region was cloned into a vector containing the gfp:lacZ reporter genes. This construct was microinjected, first into C. elegans to demonstrate promoter function, and then into both tree-living and parasitic adults of P. trichosuri. Reporter gene expression was shown in the progeny of microinjected parasitic adults. RNA was made from infective P. trichosuri larvae, reverse transcribed and the coding sequence for the PtHsp70 protein cloned into an expression vector and expressed in E. coli, The recombinant protein pattern had a similar pattern of trypsin digestion products as the native protein, as shown by MALDI-TOF mass spectrometry, but it was immunologically distinct from the native protein. The culmination of this project was the generation of a transgenic P trichosuri, the first vertebrate endoparasitic nematode to be heritably transformed. This is a necessary step in the development of a self-disseminating vector to be used in the biocontrol of possums.</p>

scholarly journals Towards the Creation of a Transgenic Possum Parasite

2021 ◽  
Author(s):  
◽  
Jan Newton-Howes
Keyword(s):  
Genomic Library ◽  
Protein Pattern ◽  
Brushtail Possum ◽  
Native Protein ◽  
Putative Promoter Region ◽  
Reading Frame ◽  
Native Flora ◽  
C Elegans ◽  
Infective Larvae ◽  
Initiator Codon

<p>The brushtail possum. Trichosurus vulpecula, is New Zealand's most serious vertebrate pest; possums destroy native flora and fauna and are vectors of bovine Tb. Conventional control is considered to be unsustainable and, in the long term, biological control is seen as the only solution to reducing possum numbers. The aim of this project is to contribute to the development of a self-disseminating vector that will spread a control molecule throughout the possum population reducing fecundity or increasing mortality. The possum-specific parasite Parastrongyloides trichosuri has considerable potential a-s such a vector. A protein from P. trichosuri specifically, was found to be antigenic in possums. The antibodies to this protein were purified from positive possum serum and used to detect the antigen on the surface of infective larvae but not in the excretory/secretory products of either larvae or adults. The protein was isolated from crude infective larvae and found to show homology to the heat-shock 70 family of proteins. Genomic DNA was extracted, an oligonucleotide probe made and a genomic library screened for the Hsp70 gene. Several positive clones were found and DNA isolated and sequenced from one such clone. Five kilo bases of unambiguous sequence was obtained in which was an open reading frame of 2 kb. Theoretical translation of this gave a protein of 64 amino acids with 80% homology to the Hsp70A protein of C. elegans. The region upstream of the ATG initiator codon was amplified and 1.3 kb of the putative promoter region was cloned into a vector containing the gfp:lacZ reporter genes. This construct was microinjected, first into C. elegans to demonstrate promoter function, and then into both tree-living and parasitic adults of P. trichosuri. Reporter gene expression was shown in the progeny of microinjected parasitic adults. RNA was made from infective P. trichosuri larvae, reverse transcribed and the coding sequence for the PtHsp70 protein cloned into an expression vector and expressed in E. coli, The recombinant protein pattern had a similar pattern of trypsin digestion products as the native protein, as shown by MALDI-TOF mass spectrometry, but it was immunologically distinct from the native protein. The culmination of this project was the generation of a transgenic P trichosuri, the first vertebrate endoparasitic nematode to be heritably transformed. This is a necessary step in the development of a self-disseminating vector to be used in the biocontrol of possums.</p>

Geminiviruses: Taxonomic Structure and Diversity in Genomic Organization

2020 ◽  
Vol 14 (2) ◽  
pp. 86-98
Author(s):  
Saurabh Kulshrestha ◽  
Abhishek Bhardwaj ◽  
Vanshika
Keyword(s):  
Transgenic Plants ◽  
Genomic Organization ◽  
Genomic Structure ◽  
Taxonomic Status ◽  
Relevant Information ◽  
Plant Viruses ◽  
Monopartite Begomoviruses ◽  
Taxonomic Structure ◽  
New Horizons ◽  
Circular Ssdna

Background: Geminiviridae is one of the best-characterized and hence, one of the largest plant-virus families with the highest economic importance. Its members characteristically have a circular ssDNA genome within the encapsidation of twinned quasi-icosaheadral virions (18-38 nm size-range). Objective: Construction of a narrative review on geminiviruses, to have a clearer picture of their genomic structure and taxonomic status. Methods: A thorough search was conducted for papers and patents regarding geminiviruses, where relevant information was used to study their genomic organization, diversity and taxonomic structure. Results: Geminiviruses have been classified into nine genera (viz., genus Begomovirus, Mastrevirus, Curtovirus, Topocuvirus, Becurtovirus, Turncurtovirus, Capulavirus, Eragrovirus and Grablovirus) having distinct genomic organizations, host ranges and insect vectors. Genomic organization of all genera generally shows the presence of 4-6 ORFs encoding for various proteins. For now, Citrus chlorotic dwarf-associated virus (CCDaV), Camellia chlorotic dwarf-associated virus (CaCDaV) and few other geminiviruses are still unassigned to any genera. The monopartite begomoviruses (and few mastreviruses) have been found associated with aplhasatellites and betasatellites (viz., ~1.3 kb circular ssDNA satellites). Recent reports suggest that deltasatellites potentially reduce the accumulation of helper-Begomovirus species in host plants. Some patents have revealed the methods to generate transgenic plants resistant to geminiviruses. Conclusion: Geminiviruses rapidly evolve and are a highly diverse group of plant-viruses. However, research has shown new horizons in tackling the acute begomoviral diseases in plants by generating a novel bio-control methodology in which deltasatellites can be used as bio-control agents and generate transgenic plants resistant to geminiviruses.

scholarly journals Genes for 2,4,5-Trichlorophenoxyacetic Acid Metabolism in Burkholderia cepacia AC1100: Characterization of the tftC and tftD Genes and Locations of the tft Operons on Multiple Replicons

1998 ◽  
Vol 64 (6) ◽  
pp. 2086-2093 ◽  
Author(s):  
Anette Hübner ◽  
Clyde E. Danganan ◽  
Luying Xun ◽  
A. M. Chakrabarty ◽  
William Hendrickson
Keyword(s):  
High Frequency ◽  
Burkholderia Cepacia ◽  
Acid Metabolism ◽  
Genomic Organization ◽  
Genomic Library ◽  
Sole Source ◽  
Amino Acid Sequences ◽  
Klebsiella Pneumonia ◽  
Trichlorophenoxyacetic Acid

ABSTRACT Burkholderia cepacia AC1100 uses the chlorinated aromatic compound 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) as a sole source of carbon and energy. The enzyme which converts the first intermediate in the pathway, 2,4,5-trichlorophenol, to 5-chlorohydroquinone has been purified and consists of two subunits of 58 and 22 kDa, encoded by the tftC and tftDgenes (48). A degenerate primer was designed from the N terminus of the 58-kDa polypeptide and used to isolate a clone containing the tftC and tftD genes from a genomic library of AC1100. The derived amino acid sequences oftftC and tftD show significant homology to the two-component monooxygenases HadA of Burkholderia pickettii, HpaBC of Escherichia coli, and HpaAH ofKlebsiella pneumonia. Expression of the tftCand tftD genes appeared to be induced when they were grown in the presence of 2,4,5-T, as shown by RNA slot blot and primer extension analyses. Three sets of cloned tft genes were used as probes to explore the genomic organization of the pathway. Pulsed-field gel electrophoresis analyses of whole chromosomes ofB. cepacia AC1100 demonstrated that the genome is comprised of five replicons of 4.0, 2.7, 0.53, 0.34, and 0.15 Mbp, designated I to V, respectively. The tft genes are located on the smaller replicons: the tftAB cluster is on replicon IV, tftEFGH is on replicon III, and copies of thetftC and the tftCD operons are found on both replicons III and IV. When cells were grown in the absence of 2,4,5-T, the genes were lost at high frequency by chromosomal deletions and rearrangements to produce 2,4,5-T-negative mutants. In one mutant, thetftA and tftB genes translocated from one replicon to another, with the concomitant loss of tftEFGHand one copy of tftCD.

Electron Probe Analysis of Vertebrate Smooth Muscle: Distribution of Ca and Ci

1976 ◽  
Vol 34 ◽  
pp. 334-335 ◽  
Author(s):  
Avril V. Somlyo ◽  
H. Shuman ◽  
A.P. Somlyo
Keyword(s):  
Smooth Muscle ◽  
Electron Probe ◽  
Preliminary Report ◽  
Cell Damage ◽  
Electron Probe Analysis ◽  
Perinuclear Space ◽  
Probe Analysis ◽  
High K ◽  
Low K ◽  
Initial Results

This is a preliminary report of electron probe analysis of rabbit portal-anterior mesenteric vein (PAMV) smooth muscle cryosectioned without fixation or cryoprotection. The instrumentation and method of electron probe quantitation used (1) and our initial results with cardiac (2) and skeletal (3) muscle have been presented elsewhere.In preparations depolarized with high K (K2SO4) solution, significant calcium peaks were detected over the sarcoplasmic reticulum (Fig 1 and 2) and the continuous perinuclear space. In some of the fibers there were also significant (up to 200 mM/kg dry wt) calcium peaks over the mitochondria. However, in smooth muscle that was not depolarized, high mitochondrial Ca was found in fibers that also contained elevated Na and low K (Fig 3). Therefore, the possibility that these Ca-loaded mitochondria are indicative of cell damage remains to be ruled out.

High resolution two-dimensional motility mapping; Methodology and initial results

2001 ◽  
Vol 120 (5) ◽  
pp. A226-A226 ◽  
Author(s):  
W LAMMERS ◽  
S DHANASEKARAN ◽  
J SLACK ◽  
B STEPHEN
Keyword(s):  
High Resolution ◽  
Two Dimensional ◽  
Initial Results
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