Differential expression of the mRNAs for the soluble and membrane-bound forms of rabbit cytochrome

Giordano, Sara J.; Steggles, Alan W.

doi:10.1016/0167-4781(93)90274-h

Cited by 19 publications

(8 citation statements)

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“…Two variants, a soluble (isoform 1) and membrane bound (isoform 2) form, of CYB5A cDNA have been isolated in pigs [18], and later detected as a low molecular weight form (isoform 1) and a high molecular weight form (isoform 2) in porcine testis [16]. The CYB5A isoforms are derived from one mRNA via alternative splicing [19]. A study by Davis et al [16] found that levels of the CYB5A isoform 1, but not levels of isoform 2, were correlated with both the rate of 16-androstene steroid synthesis and fat androstenone concentrations, indicating that increased levels of the isoform 1 of CYB5A are linked to a higher level of androstenone production in pig testis.…”

Section: Discussionmentioning

confidence: 99%

Transcript profiling of candidate genes in testis of pigs exhibiting large differences in androstenone levels

et al. 2010

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BackgroundBoar taint is an unpleasant odor and flavor of the meat and occurs in a high proportion of uncastrated male pigs. Androstenone, a steroid produced in testis and acting as a sex pheromone regulating reproductive function in female pigs, is one of the main compounds responsible for boar taint. The primary goal of the present investigation was to determine the differential gene expression of selected candidate genes related to levels of androstenone in pigs.ResultsAltogether 2560 boars from the Norwegian Landrace and Duroc populations were included in this study. Testicle samples from the 192 boars with most extreme high or low levels of androstenone in fat were used for RNA extraction, and 15 candidate genes were selected and analyzed by real-competitive PCR analysis. The genes Cytochrome P450 c17 (CYP17A1), Steroidogenic acute regulatory protein (STAR), Aldo-keto reductase family 1 member C4 (AKR1C4), Short-chain dehydrogenase/reductase family member 4 (DHRS4), Ferritin light polypeptide (FTL), Sulfotransferase family 2A, dehydroepiandrosterone-preferring member 1 (SULT2A1), Cytochrome P450 subfamily XIA polypeptide 1 (CYP11A1), Cytochrome b5 (CYB5A), and 17-beta-Hydroxysteroid dehydrogenase IV (HSD17B4) were all found to be significantly (P < 0.05) up-regulated in high androstenone boars in both Duroc and Landrace. Furthermore, Cytochrome P450 c19A2 (CYP19A2) was down-regulated and progesterone receptor membrane component 1 (PGRMC1) was up-regulated in high-androstenone Duroc boars only, while CYP21 was significantly down-regulated (2.5) in high-androstenone Landrace only. The genes Nuclear Receptor co-activator 4 (NCOA4), Sphingomyrlin phosphodiesterase 1 (SMPD1) and 3β-hydroxysteroid dehydrogenase (HSD3B) were not significantly differentially expressed in any breeds. Additionally, association studies were performed for the genes with one or more detected SNPs. Association between SNP and androstenone level was observed in CYB5A only, suggesting cis-regulation of the differential transcription in this gene.ConclusionA large pig material of highly extreme androstenone levels is investigated. The current study contributes to the knowledge about which genes that is differentially expressed regard to the levels of androstenone in pigs. Results in this paper suggest that several genes are important in the regulation of androstenone level in boars and warrant further evaluation of the above mentioned candidate genes, including analyses in different breeds, identification of causal mutations and possible gene interactions.

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Section: Discussionmentioning

confidence: 99%

Transcript profiling of candidate genes in testis of pigs exhibiting large differences in androstenone levels

et al. 2010

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“…The full amino acid sequence of rabbit cyt b 5 was aligned with 16 other cyt b 5 sequences obtained from the National Center of Biotechnology Information (NCBI) nucleotide data base using BLAST (11,12). The multiple-sequence alignment program ClustalW was used to align the heme domains and identify conserved residues (13).…”

Section: Methodsmentioning

confidence: 99%

The role of the length and sequence of the linker domain of cytochrome b5 in stimulating cytochrome P450 2B4 catalysis.

Clarke

Bidwai

et al. 2004

Journal of Biological Chemistry

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Cytochrome b 5 (cyt b 5 ) is a 15-kDa amphipathic protein with a cytosolic amino-terminal catalytic heme domain, which is anchored to the microsomal membrane by a hydrophobic transmembrane ␣-helix at its carboxyl terminus. These two domains are connected by an ϳ15-amino acid linker domain, Ser 90 -Asp 104 , which has been modified by site-directed mutagenesis to investigate whether the length or sequence of the linker influences the ability of cyt b 5 to bind ferric cytochrome P450 2B4 and donate an electron to oxyferrous (cyt P450 2B4), thereby stimulating catalysis. Because shortening the linker by 8 or more amino acids markedly inhibited the ability of cyt b 5 to bind cyt P450 2B4 and stimulate catalysis by this isozyme, it is postulated 7 amino acids are sufficient to allow a productive interaction. All mutant cyts b 5 except the protein lacking the entire 15-amino acid linker inserted normally into the microsomal membrane. Alternatively, lengthening the linker by 16 amino acids, reversing the sequence of the amino acids in the linker, and mutating conserved linker residues did not significantly alter the ability of cyt b 5 to interact with cyt P450 2B4. A model for the membranebound cyt b 5 -cyt P450 complex is presented.Microsomal cytochrome b 5 (cyt b 5 ) 1 is an amphipathic electron transfer hemoprotein located in the membrane of the endoplasmic reticulum. It provides electrons for a broad range of reactions, including fatty acid desaturation, cholesterol biosynthesis, and a variety of cytochrome P450-dependent oxidation and hydroxylation reactions (1, 2).Cytochrome P450 (cyt P450) is responsible for the oxidation of a large number of substrates. The overall stoichiometry of the reaction catalyzed by cyt P450 is shown in Reaction 1, where RH is the substrate (3).The cyt P450 catalytic reaction occurs via a reaction cycle that involves substrate binding, reduction of the ferric heme, O 2 binding, reduction of the oxyferrous heme (second electron transfer), substrate oxidation, and finally product dissociation (4). Both ferric and oxyferrous cyt P450 receive electrons from NADPH via its redox partner, NADPH-cytochrome P450 reductase (cyt P450 reductase), whereas cyt b 5 can donate the second electron. Because of the high redox potential of Х20 mV for cyt b 5 , the first electron required to reduce cyt P450 from the ferric to the ferrous state is always transferred from NADPH via cyt P450 reductase. However, as the redox potential of cyt P450 increases from ХϪ230 mV to ϩ50 mV on transition to the oxyferrous state, the second electron can be obtained from either cyt P450 reductase or cyt b 5 (5, 6).Depending on the substrate, isozyme of cyt P450 and the experimental conditions cyt b 5 can stimulate, inhibit, or have no effect on cyt P450 activity (2). Studies of the stoichiometry of the metabolism of benzphetamine and methoxyflurane by cyt P450 2B4 have shown that cyt b 5 increases the efficiency of catalysis by cyt P450 2B4 primarily by decreasing the formation of the side-product superoxide. However, cyt b...

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“…The normal adrenal gland has been previously shown to contain cytochrome b5 mRNA (Giordano and Steggles 1993). Since high levels of cytochrome b 5 were found to be associated with high levels of the lyase activity of 17c~-hydroxylase as well as high levels of androgen production in andrenocortical adenomas (Sakai et al 1994), perhaps the lack of cytochrome b5 activity in the adrenals and testes of this patient is associated with very low levels of androgen synthesis and resultant pseudohermaphrodism.…”

Section: Discussionmentioning

confidence: 94%

“…RNA was extracted from leukocytes and reticulocytes using the acid guanidinium thiocyanate-phenol method described by Chomczynski and Sacchi (1987), and modified by Giordano and Steggles (1993) for use with small volumes of frozen cells. Total RNA was reverse transcribed using random hexamers (Boehringer Mannheim, Indianapolis, Ind.)…”

Section: Synthesis Of Cdnamentioning

confidence: 99%

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A splicing mutation in the cytochrome b5 gene from a patient with congenital methemoglobinemia and pseudohermaphrodism

1994

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We have analyzed reticulocyte and leukocyte mRNAs isolated from a patient with congenital methemoglobinemia and pseudohermaphrodism. The cytochrome b5 cDNA sequences were amplified using specific oligonucleotide primers and the polymerase chain reaction (PCR). DNA sequencing indicated that there was a 16-bp deletion in the cDNA leading to a new, in-frame stop signal and resulting in a truncated protein of 45 amino acids. Genomic DNA was analyzed, and the molecular lesion was shown to be an AG-->GG alteration in the 3' splicing junction of intron 1. The splice site alteration leads to the usage of the nearest AG as an alternative splice site, resulting in a 16-bp deletion in the mRNA. All of the studies on reticulocyte mRNA and genomic DNA indicated that the patient was homozygous for the lesion.

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Differential expression of the mRNAs for the soluble and membrane-bound forms of rabbit cytochrome

Cited by 19 publications

References 30 publications

Transcript profiling of candidate genes in testis of pigs exhibiting large differences in androstenone levels

Transcript profiling of candidate genes in testis of pigs exhibiting large differences in androstenone levels

The role of the length and sequence of the linker domain of cytochrome b5 in stimulating cytochrome P450 2B4 catalysis.

A splicing mutation in the cytochrome b5 gene from a patient with congenital methemoglobinemia and pseudohermaphrodism

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