Biomaterial Database

A cytochrome-b5-containing fusion protein similar to plant acyl lipid desaturases. 1995

P Sperling, and H Schmidt, and E Heinz

Institut für Allgemeine Botanik, Universität Hamburg, Germany.

The similarity between oleate and linoleate desaturase sequences from several plants was used to construct degenerate oligonucleotide primers for PCR experiments with cDNA transcribed from mRNA of ripening sunflower embryos. A DNA fragment was amplified and sequenced. Specific primers derived from this partial sequence were used for rapid amplification of the 3'- and 5'-ends of this cDNA. With appropriate primers derived from these sequences, a full-length clone of 1377 bp was amplified by PCR which, after sequencing, showed an open reading frame of 458 amino acids corresponding to a putative protein of about 52 kDa. Comparison with other desaturases showed the conserved three histidine boxes and the characteristic hydropathy profile of membrane-bound desaturases, but the amino acid identity was restricted to 18% and the N-terminal region was about 100 amino acids longer. This N-terminal extension showed high similarity with cytochrome b5 and, accordingly, the whole sequence can be considered as coding for a fusion protein between cytochrome b5 and a desaturase-like enzyme. Furthermore, we detected a similar cytochrome b5 fold in the previously sequenced delta 9 acyl-CoA desaturase from yeast, but in this enzyme it was located at the C-terminus. An alignment of these fusion proteins with other heme-binding proteins revealed desaturases to be novel members of the cytochrome b5 superfamily. A truncated DNA representing 366 bp of the 5'-end was amplified from the cDNA clone and expressed in Escherichia coli. The truncated cDNA coded for a soluble protein of about 12 kDa as shown by SDS/PAGE and N-terminal sequencing. The enriched recombinant protein exhibited redox absorbance spectra characteristic of plant microsomal cytochrome b5.

UI	MeSH Term	Description	Entries
D008969	Molecular Sequence Data	Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories.	Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D011994	Recombinant Proteins	Proteins prepared by recombinant DNA technology.	Biosynthetic Protein,Biosynthetic Proteins,DNA Recombinant Proteins,Recombinant Protein,Proteins, Biosynthetic,Proteins, Recombinant DNA,DNA Proteins, Recombinant,Protein, Biosynthetic,Protein, Recombinant,Proteins, DNA Recombinant,Proteins, Recombinant,Recombinant DNA Proteins,Recombinant Proteins, DNA
D003001	Cloning, Molecular	The insertion of recombinant DNA molecules from prokaryotic and/or eukaryotic sources into a replicating vehicle, such as a plasmid or virus vector, and the introduction of the resultant hybrid molecules into recipient cells without altering the viability of those cells.	Molecular Cloning
D004926	Escherichia coli	A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc.	Alkalescens-Dispar Group,Bacillus coli,Bacterium coli,Bacterium coli commune,Diffusely Adherent Escherichia coli,E coli,EAggEC,Enteroaggregative Escherichia coli,Enterococcus coli,Diffusely Adherent E. coli,Enteroaggregative E. coli,Enteroinvasive E. coli,Enteroinvasive Escherichia coli
D006368	Helianthus	A genus herbs of the Asteraceae family. The SEEDS yield oil and are used as food and animal feed; the roots of Helianthus tuberosum (Jerusalem artichoke) are edible.	Jerusalem Artichoke,Sunflower,Helianthus annuus,Helianthus tuberosus,Artichoke, Jerusalem,Sunflowers
D000595	Amino Acid Sequence	The order of amino acids as they occur in a polypeptide chain. This is referred to as the primary structure of proteins. It is of fundamental importance in determining PROTEIN CONFORMATION.	Protein Structure, Primary,Amino Acid Sequences,Sequence, Amino Acid,Sequences, Amino Acid,Primary Protein Structure,Primary Protein Structures,Protein Structures, Primary,Structure, Primary Protein,Structures, Primary Protein
D001483	Base Sequence	The sequence of PURINES and PYRIMIDINES in nucleic acids and polynucleotides. It is also called nucleotide sequence.	DNA Sequence,Nucleotide Sequence,RNA Sequence,DNA Sequences,Base Sequences,Nucleotide Sequences,RNA Sequences,Sequence, Base,Sequence, DNA,Sequence, Nucleotide,Sequence, RNA,Sequences, Base,Sequences, DNA,Sequences, Nucleotide,Sequences, RNA
D012639	Seeds	The encapsulated embryos of flowering plants. They are used as is or for animal feed because of the high content of concentrated nutrients like starches, proteins, and fats. Rapeseed, cottonseed, and sunflower seed are also produced for the oils (fats) they yield.	Diaspores,Elaiosomes,Embryos, Plant,Plant Embryos,Plant Zygotes,Zygotes, Plant,Diaspore,Elaiosome,Embryo, Plant,Plant Embryo,Plant Zygote,Seed,Zygote, Plant
D013053	Spectrophotometry	The art or process of comparing photometrically the relative intensities of the light in different parts of the spectrum.
D015786	Cytochromes b5	Cytochromes of the b group that are found bound to cytoplasmic side of ENDOPLASMIC RETICULUM. They serve as electron carrier proteins for a variety of membrane-bound OXYGENASES. They are reduced by the enzyme CYTOCHROME-B(5) REDUCTASE.	Apocytochrome b5,Cytochrome b-5,Cytochrome b5,Ferricytochrome b5,Cytochrome b 5