scholarly journals Characterization of an Arabidopsis inositol 1,3,4,5,6-pentakisphosphate 2-kinase (AtIPK1)

2006 ◽  
Vol 394 (1) ◽  
pp. 95-103 ◽  
Author(s):  
Dylan Sweetman ◽  
Sue Johnson ◽  
Samuel E. K. Caddick ◽  
David E. Hanke ◽  
Charles A. Brearley
Keyword(s):  
Mutant Strain ◽  
Genomic Sequence ◽  
Inositol Phosphate ◽  
Amino Acid Sequences ◽  
Pcr Analysis ◽  
Temperature Sensitive ◽  
Reading Frame ◽  
Human Orthologue

The metabolic pathway(s) by which plants synthesize InsP6 (inositol 1,2,3,4,5,6-hexakisphosphate) remains largely undefined [Shears (1998) Biochim. Biophys. Acta 1436, 49–67], while the identities of the genes that encode enzymes catalysing individual steps in these pathways are, with the notable exception of myo-inositol phosphate synthase and ZmIpk [Shi, Wang, Wu, Hazebroek, Meeley and Ertl (2003) Plant Physiol. 131, 507–515], unidentified. A yeast enzyme, ScIPK1, catalyses the synthesis of InsP6 by 2-phosphorylation of Ins(1,3,4,5,6)P5 (inositol 1,3,4,5,6-pentakisphosphate). A human orthologue, HsIPK1, is able to substitute for yeast ScIPK1, restoring InsP6 production in a Saccharomyces cerevisiae mutant strain lacking the ScIPK1 open reading frame (ScIpk1Δ). We have identified an Arabidopsis genomic sequence, AtIPK1, encoding an Ins(1,3,4,5,6)P5 2-kinase. Inclusion of the AtIPK1 protein in alignments of amino acid sequences reveals that human and Arabidopis kinases are more similar to each other than to the S. cerevisiae enzyme, and further identifies an additional motif. Recombinant AtIPK1 protein expressed in Escherichia coli catalysed the synthesis of InsP6 from Ins(1,3,4,5,6)P5. The enzyme obeyed Michaelis–Menten kinetics with an apparent Vmax of 35 nmol·min−1·(mg of protein)−1 and a Km for Ins(1,3,4,5,6)P5 of 22 μM at 0.4 mM ATP. RT (reverse transcriptase)–PCR analysis of AtIPK1 transcripts revealed that AtIPK1 is expressed in siliques, leaves and cauline leaves. In situ hybridization experiments further revealed strong expression of AtIPK1 in male and female organs of flower buds. Expression of AtIPK1 protein in an ScIpk1Δ mutant strain restored InsP6 production and rescued the temperature-sensitive growth phenotype of the yeast.

scholarly journals Identification and Characterization of thefis Operon in Enteric Bacteria

1998 ◽  
Vol 180 (22) ◽  
pp. 5932-5946 ◽  
Author(s):  
Michael B. Beach ◽  
Robert Osuna
Keyword(s):  
Amino Acid ◽  
Genomic Sequence ◽  
Erwinia Carotovora ◽  
Enteric Bacteria ◽  
Integration Host Factor ◽  
Amino Acid Sequences ◽  
Reading Frame ◽  
E Coli

ABSTRACT The small DNA binding protein Fis is involved in several different biological processes in Escherichia coli. It has been shown to stimulate DNA inversion reactions mediated by the Hin family of recombinases, stimulate integration and excision of phage λ genome, regulate the transcription of several different genes including those of stable RNA operons, and regulate the initiation of DNA replication at oriC. fis has also been isolated from Salmonella typhimurium, and the genomic sequence of Haemophilus influenzae reveals its presence in this bacteria. This work extends the characterization of fis to other organisms. Very similar fis operon structures were identified in the enteric bacteria Klebsiella pneumoniae, Serratia marcescens, Erwinia carotovora, andProteus vulgaris but not in several nonenteric bacteria. We found that the deduced amino acid sequences for Fis are 100% identical in K. pneumoniae, S. marcescens,E. coli, and S. typhimurium and 96 to 98% identical when E. carotovora and P. vulgaris Fis are considered. The deduced amino acid sequence forH. influenzae Fis is about 80% identical and 90% similar to Fis in enteric bacteria. However, in spite of these similarities, the E. carotovora, P. vulgaris, and H. influenzae Fis proteins are not functionally identical. An open reading frame (ORF1) precedingfis in E. coli is also found in all these bacteria, and their deduced amino acid sequences are also very similar. The sequence preceding ORF1 in the enteric bacteria showed a very strong similarity to the E. coli fis P region from −53 to +27 and the region around −116 containing an ihfbinding site. Both β-galactosidase assays and primer extension assays showed that these regions function as promoters in vivo and are subject to growth phase-dependent regulation. However, their promoter strengths vary, as do their responses to Fis autoregulation and integration host factor stimulation.

scholarly journals Analysis of The Open Reading Frame (ORF) 29-TrnC (GCA) Sequence to Detect Indica and Japonica Sub Species on Upland Rice of Situ Bagendit and Inbred Rice of Ciherang

2018 ◽  
Vol 10 (1) ◽  
pp. 153-159
Author(s):  
Rohma Istiana ◽  
Hermin Pancasakti Kusumaningrum ◽  
Rejeki Siti Ferniah
Keyword(s):  
Plant Breeding ◽  
Upland Rice ◽  
Pcr Amplification ◽  
Breeding Program ◽  
Abiotic Factor ◽  
Reading Frame ◽  
Plant Breeding Program ◽  
Pcr Products

The identification and the characterization of genetic diversity of rice was the first step in the rice plant breeding program. This study aimed to detect indica or japonica sub-species on upland rice Situ Bagendit and inbred rice Ciherang using molecular markers ORF 29-TrnC (GCA) on the chloroplast genome. Rice was included to the indica sub-species if the 32 bp insertion on ORF 29-TrnC (GCA) sequence was found, on the contrary, if the deletion 32 bp on ORF 29-TrnC (GCA) was found then it was included to the japonica sub-species. DNA isolation was examined from the leaves of the rice plants, and then it tested quantitatively to determine the transparency and DNA concentration from the isolation results. PCR amplification was performed using a pair of primers CP2 and it was followed by agarose gel electrophoresis. The visualization of the DNA bands used the gel documentation. Sequencing of PCR products produced a long base 390 bp in Situ Bagendit rice and 390 bp in Ciherang rice. Analysis of the sequences showed that the insertions occurred throughout the 32 bp in Situ Bagendit rice and the insertions occurred throughout the 32 bp in Ciherang rice. The results showed that upland rice Situ Bagendit and inbred rice Ciherang were included in the indica sub-species. The knowledge of variety of genetics of rice can be used as bio-information in the plant breeding program. Further, the knowledge can be used to protect in genetic power source, the selection and the composing of superior varieties of rice which is tolerant with kinds of biotic and abiotic factor.

scholarly journals Molecular Characterization of a Novel Cathepsin L in Macrobrachium nipponense and Its Function in Ovary Maturation

2022 ◽  
Vol 12 ◽  
Author(s):  
Sufei Jiang ◽  
Yiwei Xiong ◽  
Wenyi Zhang ◽  
Junpeng Zhu ◽  
Dan Cheng ◽  
...  
Keyword(s):  
Cathepsin L ◽  
Cysteine Proteases ◽  
Macrobrachium Nipponense ◽  
Reading Frame ◽  
Physiological Processes ◽  
Gonad Somatic Index ◽  
Oriental River Prawn ◽  
Ovary Maturation

Cathepsin L genes, which belonged to cysteine proteases, were a series of multifunctional protease and played important roles in a lot of pathological and physiological processes. In this study, we analyzed the characteristics a cathepsin L (named Mn-CL2) in the female oriental river prawn, Macrobrachium nipponense which was involved in ovary maturation. The Mn-CL2 was1,582 bp in length, including a 978 bp open reading frame that encoded 326 amino acids. The Mn-CL2 was classified into the cathepsin L group by phylogenetic analysis. Real-time PCR (qPCR) analysis indicated that Mn-CL2 was highly expressed in the hepatopancreas and ovaries of female prawns. During the different ovarian stages, Mn-CL2 expression in the hepatopancreas and ovaries peaked before ovarian maturation. In situ hybridization studies revealed that Mn-CL2 was localized in the oocyte of the ovary. Injection of Mn-CL2 dsRNA significantly reduced the expression of vitellogenin. Changes in the gonad somatic index also confirmed the inhibitory effects of Mn-CL2 dsRNA on ovary maturation. These results suggest that Mn-CL2 has a key role in promoting ovary maturation.

Identification and preliminary characterization of cell-wall-anchored proteins of Staphylococcus epidermidis

Microbiology ◽  
2005 ◽  
Vol 151 (5) ◽  
pp. 1453-1464 ◽  
Author(s):  
M. Gabriela Bowden ◽  
Wei Chen ◽  
Jenny Singvall ◽  
Yi Xu ◽  
Sharon J. Peacock ◽  
...  
Keyword(s):  
Cell Wall ◽  
Staphylococcus Epidermidis ◽  
Tertiary Structure ◽  
Genomic Sequence ◽  
Human Infection ◽  
Pcr Analysis ◽  
Genes Encoding ◽  
Antibody Titres

Staphylococcus epidermidis is a ubiquitous human skin commensal that has emerged as a major cause of foreign-body infections. Eleven genes encoding putative cell-wall-anchored proteins were identified by computer analysis of the publicly available S. epidermidis unfinished genomic sequence. Four genes encode previously described proteins (Aap, Bhp, SdrF and SdrG), while the remaining seven have not been characterized. Analysis of primary sequences of the Staphylococcus epidermidis surface (Ses) proteins indicates that they have a structural organization similar to the previously described cell-wall-anchored proteins from S. aureus and other Gram-positive cocci. However, not all of the Ses proteins are direct homologues of the S. aureus proteins. Secondary and tertiary structure predictions suggest that most of the Ses proteins are composed of several contiguous subdomains, and that the majority of these predicted subdomains are folded into β-rich structures. PCR analysis indicates that certain genes may be found more frequently in disease isolates compared to strains isolated from healthy skin. Patients recovering from S. epidermidis infections had higher antibody titres against some Ses proteins, implying that these proteins are expressed during human infection. Western blot analyses of early-logarithmic and late-stationary in vitro cultures suggest that different regulatory mechanisms control the expression of the Ses proteins.

Organization and expression of the chicken N-myc gene

1990 ◽  
Vol 10 (5) ◽  
pp. 2017-2026
Author(s):  
S Sawai ◽  
K Kato ◽  
Y Wakamatsu ◽  
H Kondoh
Keyword(s):  
Genomic Sequence ◽  
Amino Acid Sequences ◽  
Coding Regions ◽  
Noncoding Exon ◽  
Exon 1 ◽  
Myc Gene ◽  
The Central Nervous System ◽  
Flanking Region ◽  
High Level

We cloned the chicken N-myc gene and analyzed its structure and expression. We found that it consisted of three exons with coding regions in exons 2 and 3. Comparison to mammalian N-myc genomic sequence indicated that nucleotide sequences of the 5'-flanking region, noncoding exon 1, and introns were not conserved, but coding and 3' noncoding sequences showed significant homology to mammalian N-myc. Alignment of deduced amino acid sequences of chicken and mammalian N-myc proteins revealed nine conserved domains interrupted by different lengths of nonhomologous sequences. Two of the domains were specific to N-myc proteins, and the other seven were common to c-myc proteins. Northern blot (immunoblot) and in situ hybridization analyses of 3.5-day-old chicken embryos revealed that high-level expression of the N-myc gene was confirmed to certain tissues, e.g., the central nervous system, neural crest derivatives, and mesenchyme of limb buds. In the beak and limb primordia, N-myc expression in the mesenchyme was higher toward the distal end, suggesting possible involvement in positional assignment of the tissue within the rudimentary structures.

scholarly journals Molecular and Insecticidal Characterization of a Cry1I Protein Toxic to Insects of the Families Noctuidae, Tortricidae, Plutellidae, and Chrysomelidae

2006 ◽  
Vol 72 (7) ◽  
pp. 4796-4804 ◽  
Author(s):  
Iñigo Ruiz de Escudero ◽  
Anna Estela ◽  
Manuel Porcar ◽  
Clara Martínez ◽  
José A. Oguiza ◽  
...  
Keyword(s):  
Specific Activity ◽  
Membrane Vesicles ◽  
Lobesia Botrana ◽  
Water Soluble ◽  
Insect Species ◽  
Pcr Analysis ◽  
Cry Protein ◽  
Toxic Activity ◽  
Reading Frame

ABSTRACT The most notable characteristic of Bacillus thuringiensis is its ability to produce insecticidal proteins. More than 300 different proteins have been described with specific activity against insect species. We report the molecular and insecticidal characterization of a novel cry gene encoding a protein of the Cry1I group with toxic activity towards insects of the families Noctuidae, Tortricidae, Plutellidae, and Chrysomelidae. PCR analysis detected a DNA sequence with an open reading frame of 2.2 kb which encodes a protein with a molecular mass of 80.9 kDa. Trypsin digestion of this protein resulted in a fragment of ca. 60 kDa, typical of activated Cry1 proteins. The deduced sequence of the protein has homologies of 96.1% with Cry1Ia1, 92.8% with Cry1Ib1, and 89.6% with Cry1Ic1. According to the Cry protein classification criteria, this protein was named Cry1Ia7. The expression of the gene in Escherichia coli resulted in a protein that was water soluble and toxic to several insect species. The 50% lethal concentrations for larvae of Earias insulana, Lobesia botrana, Plutella xylostella, and Leptinotarsa decemlineata were 21.1, 8.6, 12.3, and 10.0 μg/ml, respectively. Binding assays with biotinylated toxins to E. insulana and L. botrana midgut membrane vesicles revealed that Cry1Ia7 does not share binding sites with Cry1Ab or Cry1Ac proteins, which are commonly present in B. thuringiensis-treated crops and commercial B. thuringiensis-based bioinsecticides. We discuss the potential of Cry1Ia7 as an active ingredient which can be used in combination with Cry1Ab or Cry1Ac in pest control and the management of resistance to B. thuringiensis toxins.

scholarly journals The identification and characterization of a testis-specific cDNA during spermatogenesis

2006 ◽  
Vol 11 (1) ◽  
Author(s):  
Ying Chen ◽  
Jiarui Hu ◽  
Ping Song ◽  
Wuming Gong
Keyword(s):  
Experimental Validation ◽  
Rna Binding ◽  
Pcr Analysis ◽  
Splicing Factors ◽  
Rt Pcr ◽  
Rich Domain ◽  
Pachytene Spermatocytes ◽  
Identification And Characterization

AbstractUsing bioinformatics and experimental validation, we obtained a cDNA (named srsf) which was exclusively expressed in the mouse testes. RT-PCR analysis showed that srsf mRNA was not expressed in the gonad during the sex determination period or during embryogenesis. In developing mouse tests, srsf expression was first detected on post-natal day 10, reached its highest level on day 23, and then reduced to and remained at a moderate level throughout adulthood. In situ hybridization analysis demonstrated that srsf mRNA was expressed in pachytene spermatocytes and round spermatids in the testes. The predicted protein contains one RNA-binding domain (RBD) and a serine-arginine rich domain (RS), which are characterized by some splicing factors of SR family members. These findings indicate that srsf may play a role during spermatogenesis.

Synthesis and Characterization of Temperature-Sensitive TSPU/PNIPAAm Semi-IPN Polymer

2011 ◽  
Vol 399-401 ◽  
pp. 359-362 ◽  
Author(s):  
Yi Chun Wang ◽  
Zheng Wei Dai ◽  
Yuan Xue
Keyword(s):  
Chemical Structure ◽  
In Situ Polymerization ◽  
Phenyl Isocyanate ◽  
Chain Extender ◽  
Synthesis And Characterization ◽  
Temperature Sensitive ◽  
Sensitive Material ◽  
Ft Ir

Thermo-sensitive polyurethane (TSPU) was synthesized from poly(ε-caprolactone) and 4,4’-methylenebis (phenyl isocyanate) by a two-step process with 1,4-butanediol as the chain extender. Following that, a novel temperature-sensitive material was created by the strategy of IPN from TSPU and PNIPAAm in the method of in situ polymerization. The chemical structure and thermo properties of the semi-IPN were characterized with FT-IR and DSC. The results prove that intensive inter-molecular interactions exist between TSPU and PNIPAAm chains, which have significant influence on the phase transition behaviors of the material. According to these results, the transition temperature of the semi-IPN can be adjusted in the range of 30~40 °C by controlling the composition of TSPU and the semi-IPNs.

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