Search results for "Cloning"
showing 10 items of 498 documents
Molecular cloning and characterization of aCandida albicansgene (EFB1) coding for the elongation factor EF-1β
1996
A Candida albicans gene homologous to Saccharomyces cerevisiae elongation factor 1 beta was isolated by screening a genomic DNA library using a C. albicans cDNA as a probe. This cDNA was previously obtained by immunoscreening of an expression library with polyclonal antibodies raised against candidal cell wall components. Sequence analysis of the cDNA and the whole C. albicans gene (EMBL accession number X96517) revealed an intron-interrupted open reading frame of 639 base pairs that encodes a 213 amino acid protein. Exon sequences are highly homologous (74%) to S. cerevisiae EFB1, whereas intron sequence is less conserved (34% identity), and the predicted amino acid sequence shares about 7…
Arbutin synthase, a novel member of the NRD1β glycosyltransferase family, is a unique multifunctional enzyme converting various natural products and …
2002
Plant glucosyltransferases (GTs) play a crucial role in natural product biosynthesis and metabolization of xenobiotics. We expressed the arbutin synthase (AS) cDNA from Rauvolfia serpentina cell suspension cultures in Escherichia coli with a 6 x His tag and purified the active enzyme to homogeneity. The recombinant enzyme had a temperature optimum of 50 degrees C and showed two different pH optima (4.5 and 6.8 or 7.5, depending on the buffer). Out of 74 natural and synthetic phenols and two cinnamyl alcohols tested as substrates for the AS, 45 were accepted, covering a broad range of structural features. Converting rates comparable to hydroquinone were not achieved. In contrast to this broa…
Acetyltransfer in natural product biosynthesis--functional cloning and molecular analysis of vinorine synthase.
2004
Vinorine synthase (EC 2.3.1.160) catalyses the acetyl-CoA- or CoA-dependent reversible formation of the alkaloids vinorine (or 11-methoxy-vinorine) and 16-epi-vellosimine (or gardneral). The forward reaction leads to vinorine, which is a direct biosynthetic precursor along the complex pathway to the monoterpenoid indole alkaloid ajmaline, an antiarrhythmic drug from the Indian medicinal plant Rauvolfia serpentina. Based on partial peptide sequences a cDNA clone was isolated and functionally expressed in Escherichia coli. The Km values of the native enzyme for gardneral and acetyl-CoA were determined to be 7.5 and 57 microM. The amino acid sequence of vinorine synthase has highest level of i…
Isolation and characterization of five Fox (Forkhead) genes from the sponge Suberites domuncula.
2003
Fox or Forkhead genes constitute a subgroup of the helix-turn-helix class of transcription factors with a characteristic and highly conserved DNA binding domain. To date, around 100 different Fox genes have been reported ranging from yeast to humans; these have been classified into 18 subclasses (A to P). Fox proteins are responsible for a wide range of functions and key roles in early developmental processes, during organogenesis and also for the function of the major organs and tissues in the adult. Here, we report the isolation and phylogenetic characterization of five members of the Fox family from the sponge Suberites domuncula. Four of them (Sd-FoxL2, Sd-FoxP, Sd-FoxD and Sd-FoxF) fal…
Characterization of a cDNA encoding RP43, a CUB-domain-containing protein from the tube of Riftia pachyptila (Vestimentifera), and distribution of it…
2000
A major 43kDa protein from the protective tube of Riftiapachyptila (Vestimentifera), named RP43, was partly microsequenced after isolation by SDS/PAGE from the protein fraction of tubes collected around the hydrothermal vents at the East Pacific Rise. On the basis of the partial peptide sequences obtained, experiments using reverse-transcriptase-mediated PCR and rapid amplification of cDNA ends led to the complete cDNA sequence. Analysis of deduced amino acid sequence of RP43 showed the presence of CUB domains (100–110-residue-spanning domains first reported in the complement subcomponents C1r/C1s, epidermal-growth-factor-related sea urchin protein and bone morphogenetic protein 1) that se…
Molecular cloning of rat G-protein-coupled receptor kinase 6 (GRK6) from brain tissue, and its mRNA expression in different brain regions and periphe…
1997
The rat G-protein-coupled receptor kinase 6 (GRK6) cDNA was cloned from rat brain tissue by a combination of reverse-transcription polymerase chain reactions (RT-PCR), based on homology to the cloned human GRK6, and rapid amplification of cDNA ends (RACE-PCR). We obtained a clone of 2817 bp with an open reading frame of 1731 bp encoding for a protein of 576 amino acids that is 96.7% identical and 97.9% similar to its human counterpart. mRNA was detectable in all brain areas examined. In addition, GRK6 was expressed in skeletal muscle, small intestine, aorta, liver, heart, lung, thymus, stomach, uterus and kidney.
Cloning, structure, cellular localization, and possible function of the tumor suppressor gene lethal(3)malignant blood neoplasm-1 of Drosophila melan…
1994
The tumor suppressor gene, lethal(3)malignant blood neoplasm-1+, of Drosophila melanogaster is required for the differentiation of the phagocytic blood-cell type, the plasmatocyte. In the homozygously mutated state it causes the malignant transformation of these blood cells. We present here the cloning, sequencing, structure, and expression of the l(3)mbn-1+ gene during development. The cloned gene was identified by germ-line transformation, generation of revertants, and the detection of the corresponding mRNA in blood cells and other tissues. Homologies of the G-S-rich C-terminus of the putative MBN83 protein to human cytokeratins K1, K10, and mouse loricrin were found. The structure and p…
A heterochromatic P sequence in the D. subobscura genome.
1994
The study of a heterochromatic P sequence of D. subobscura reveals that it is a degraded element, located at the centromeric region of the A chromosome (X chromosome in this species), and that it is strongly diverged from the euchromatic P sequences previously described in this species. This heterochromatic sequence is composed of some P element fragments embedded in undefined beta-heterochromatic sequences. These mosaic P sequences do not show any transcriptional activity and seem to be ancient parasites of the D. subobscura genome. Phylogenetic analyses indicate that both the euchromatic and heterochromatic P sequences of D. subobscura could come from an ancestral element which was presen…
Src proteins/src genes: from sponges to mammals
2004
The genome of marine sponge Suberites domuncula, a member of the most ancient and most simple metazoan phylum Porifera, encodes at least five genes for Src-type proteins, more than, i.e., Caenorhabditis elegans or Drosophila melanogaster (two in each). Three proteins, SRC1SD, SRC2SD and SRC3SD, were fully characterized. The overall homology (identity+similarity) among the three S. domuncula Srcs (68-71%) is much lower than the sequence conservation between orthologous Src proteins from freshwater sponges (82-85%). It is therefore very likely that several src genes/proteins were already present in the genome of Urmetazoa, the hypothetical metazoan ancestor. We have identified in the S. domun…
Cloning and Sequencing of a cDNA Encoding a Larval-Pupal-Specific Cuticular Protein in Tenebrio Molitor (Insecta, Coleoptera). Developmental Expressi…
1996
A cDNA clone encoding a larval-pupal cuticular protein, named TMLPCP-22, has been isolated by screening a library in expression vector with a monoclonal antibody made against pupal cuticular proteins of Tenebrio molitor. Northern-blot and in situ hybridization analyses showed that the expression of TMLPCP-22 is regulated in a stage-specific and tissue-specific manner; the transcript was present during the secretion of preecdysial larval and pupal cuticles and was restricted to epidermal cells. No expression was observed during adult cuticle deposition. In supernumerary pupae obtained after application of a juvenile hormone analogue, which is known to inhibit the adult programme, TMLPCP-22 m…