Comparative sequence analysis of the PRKAG3 region between human and pig: evolution of repetitive sequences and potential new exons

The PRKAG3 gene encodes the gamma3 chain of AMP-activated protein kinase (AMPK). A non-conservative missense mutation in the PRKAG3 gene causes a dominant phenotype involving abnormally high glycogen content in pig skeletal muscle. We have determined >126 kb (in 13 contigs) of porcine genomic sequence surrounding the PRKAG3 gene and the corresponding mouse region covering the gene. A comparison of these PRKAG3 sequences and the human sequence was conducted and used to predict evolutionarily conserved regions, including regulatory regions. A comparison of the human genomic sequence and a porcine BAC sequence containing the PRKAG3 gene, revealed a conserved organization and the presence of three additional genes, CYP27A1 (cytochrome P450, family 27, subfamily A, polypeptide 1), STK36 (Serine Threonine Kinase 36), and the homolog of the unidentified human mRNA KIAA0173. Interspersed repetitive elements constituted 51.4 and 38.6% of this genomic region in human and pig, respectively. We were able to reliably align 12.6 kb of orthologous repeats shared between pig and human and these showed an average sequence identity of 72.4%. Our analysis revealed that the human KIAA0173 gene harbors alternative 5' untranslated exons originating from repetitive elements. This provides an obvious example how transposable elements may affect gene evolution.     

家蚕P450基因CYP18A1的克隆、序列分析及转录活性

蜕皮激素对昆虫生长、发育和繁殖有重要调控作用,尤其对蜕皮和变态过程。利用GenBank上登录的蜕皮激素C₂₆羟基化酶候选基因CYP18A1的氨基酸序列对家蚕Bombyx mori全基因组数据库进行BLASTP比对,发现了家蚕直向同源基因(ortholog),其完全编码序列经RT-PCR检测和克隆、测序验证后,再以此为信息探针检索家蚕表达序列标签(expressed sequence tags,EST)数据库进行拼接延伸,获得了一条包括5′非翻译区在内的长度为1 737 bp的cDNA序列,验证结果也表明与电子克隆序列完全一致(GenBank登录号为EF421988,P450命名委员会将其命名为CYP18A1)。该基因的开放阅读框为1 623 bp,编码541个氨基酸,含有包括P450s特征结构域在内的所有昆虫P450基因的5个保守结构域,其推定的分子量为61.67 kD,等电点为 8.54。将该基因cDNA序列与家蚕基因组序列进行比对,结果表明该基因具有6个外显子,5个内含子,外显子／内含子边界符合经典的GT-AG规则。同源性分析也发现家蚕CYP18A1与其他昆虫的直向同源基因具有较高相似性。用RT-PCR方法对家蚕主要发育变态时期与组织进行检测,显示出该基因的转录表达不仅具有时空特异性,而且在表达时期上与已报道的蚕体内蜕皮激素含量变化有紧密的一致性。该研究进一步证实了CYP18A1基因与昆虫体内蜕皮激素代谢平衡相关联。     

Diversity and distribution of CYP gene family in Bactrian camel

Cytochrome P450 (CYP) enzymes belong to a superfamily of monooxygenases which are phase I enzymes responsible for the first pass metabolism of about 90% of drugs in animals. However, these enzymes are often polymorphic and metabolism of the same drug in different species or different individuals is influenced by genetic and non-genetic factors. Bactrian camels are capable of survival in harsh living environments, being able to consume diets that are often toxic to other mammals and can tolerate extreme water and food deprivation. The aim of this study was to investigate whether the Bactrian camel’s special metabolic pathways and unique detoxification capabilities are attributable to particularities of the CYP gene family. The Bactrian camel’s whole genome sequencing data were systemically analyzed and annotated, and then, CYP gene family was searched from the whole protein database and compared with CYP gene families of cattle, horse, chicken, and human. The total of 63 CYP gene copies were found in Bactrian camel’s whole genome and were classified into 17 families and 38 subfamilies. Among them, 9 multi-gene families were found, and CYP2, CYP3, and CPY4 have 27, 6, and 7 subfamilies, accounting for 43, 10, and 11% in camel CYP gene, respectively. In comparison with cattle, chicken, horse, and human, the distribution of CYP gene subfamilies in camel is different, with more CYP2J and CYP3A copies in the Bactrian camel, which may contribute to the Bactrian camel’s specific biological characteristics and metabolic pathways. Comparing to the cow, horse, chicken, and human CYP genes, the distribution of CYP gene subfamilies is distinct in the Bactrian camel. The higher copy number of CYP2J gene and CYP3A gene in Bactrian camel may be the important factors contributing to the distinct biological characteristics and metabolic pathways of Bactrian camels for adaptation to the harsh environments.     

Molecular evolution of the insect Halloween family of cytochrome P450s: phylogeny, gene organization and functional conservation

The insect molting hormone, 20-hydroxyecdysone (20E), is a major modulator of the developmental processes resulting in molting and metamorphosis. During evolution selective forces have preserved the Halloween genes encoding cytochrome P450 (P450) enzymes that mediate the biosynthesis of 20E. In the present study, we examine the phylogenetic relationships of these P450 genes in holometabolous insects belonging to the orders Hymenoptera, Coleoptera, Lepidoptera and Diptera. The analyzed insect genomes each contains single orthologs of Phantom (CYP306A1), Disembodied (CYP302A1), Shadow (CYP315A1) and Shade (CYP314A1), the terminal hydroxylases. In Drosophila melanogaster, the Halloween gene spook (Cyp307a1) is required for the biosynthesis of 20E, although a function has not yet been identified. Unlike the other Halloween genes, the ancestor of this gene evolved into three paralogs, all in the CYP307 family, through gene duplication. The genomic stability of these paralogs varies among species. Intron-exon structures indicate that D. melanogaster Cyp307a1 is a mRNA-derived paralog of spookier (Cyp307a2), which is the ancestral gene and the closest ortholog of the coleopteran, lepidopteran and mosquito CYP307A subfamily genes. Evolutionary links between the insect Halloween genes and vertebrate steroidogenic P450s suggest that they originated from common ancestors, perhaps destined for steroidogenesis, before the deuterostome-arthropod split. Conservation of putative substrate recognition sites of orthologous Halloween genes indicates selective constraint on these residues to prevent functional divergence. The results suggest that duplications of ancestral P450 genes that acquired novel functions may have been an important mechanism for evolving the ecdysteroidogenic pathway.     

Genome analyses of Streptomyces peucetius ATCC 27952 for the identification and comparison of cytochrome P450 complement with other Streptomyces

We have determined the genome sequence of 8.7 Mb chromosome of Streptomyces peucetius ATCC 27952, which produces clinically important anthracycline chemotherapeutic agents of the polyketide class of antibiotics, daunorubicin and doxorubicin. The cytochrome P450 (CYP) superfamily is represented by 19 sequences in the S. peucetius. Among those, 15 code for functional genes, whereas the remaining four are pseudo genes. CYPs from S. peucetius are phylogenetically close to those of Streptomyces amermitilis. Four CYPs are associated with modular PKS of avermectin and two with doxorubicin biosynthetic gene cluster. CYP252A1 is the new family found in S. peucetius, which shares 38% identity to CYP51 from Streptomyces coelicolor A3 (2). Nine CYPs from S. peucetius are found in the cluster containing various regulatory genes including rar operon, conserved in S. coelicolor A3 (2) and Streptomyces griseus. Although two ferredoxins and four ferredoxin reductases have been identified so far, only one ferredoxin reductase was found in the cluster of CYP147F1 in S. peucetius. To date, 174 CYPs have been described from 45 Streptomyces species in all searchable databases. However, only 18 CYPs are clustered with ferredoxin. The comparative study of cytochrome P450s, ferredoxins, and ferredoxin reductases should be useful for the future development and manipulation of antibiotic biosynthetic pathways.     

CYP3A4 and pregnane X receptor humanized mice

Marked species differences exist in P450 expression and activities. In order to produce mouse models that can be used to more accurately predict human drug and carcinogen metabolism, P450- and xenobiotic receptor humanized mice are being prepared using bacterial artificial chromosomes (BAC) and P1 phage artificial chromosomes (PAC) genomic clones. In some cases, transgenic mice carrying the human genes are bred with null-mice to produce fully humanized mice. Mice expressing human CYP1A1, CYP1A2, CYP2E1, CYP2D6, CYP3A4, and CYP3A7 were generated and characterized. Studies with the CYP3A4-humanized (hCYP3A4) mouse line revealed new information on the physiological function of this P450 and its role in drug metabolism in vivo. With this mouse line, CYP3A4, under certain circumstances, was found to alter the serum levels of estrogen resulting in deficient lactation and low pup survival as a result of underdeveloped mammary glands. This hCYP3A4 mouse established the importance of intestinal CYP3A4 in the pharmacokinetics of orally administered drugs. The hCYP3A4 mice were also used to establish the mechanisms of potential gender differences in CYP3A4 expression (adult female > adult male) that could account for human gender differences in drug metabolism and response. The pregnane X receptor (PXR) is also involved in induction of drug metabolism through its target genes including CYP3A4. Since species differences exist in ligand specificity between human and mice, a PXR-humanized mouse (hPXR) was produced that responds to human PXR activators such as rifampicin but does not respond to the rodent activator pregnenalone 16alpha-carbonitrile.     

Equine cytochrome P450 2C92: cDNA cloning, expression and initial characterization

Substantial gaps exist in our knowledge of the metabolic clearance of therapeutic agents in horses. Accordingly, a cytochrome P450 monooxygenase in the 2C family was cloned from an equine liver, sequenced and expressed in a baculovirus expression system. Catalytic activities of the recombinant protein were measured with a number of substrates. The protein, assigned CYP2C92, displayed optimal catalytic activity with diclofenac using molar ratios of CYP2C92 to NADPH CYP450 reductase of 1:18. Addition of cytochrome b₅ to diclofenac incubations had no significant effect on metabolic turnover. CYP2C92 catalyzed diclofenac metabolism was 20-fold slower than the human counterpart, CYP2C9. CYP2C92 demonstrated comparable tolbutamide and (S)-warfarin hydroxylase activity compared to CYP2C9, upon addition of b₅ to the reactions. The results of this study demonstrate substantial interspecies differences in metabolism of substrates by CYP2C orthologues in the horse and human and support the need to fully characterize this enzyme system in equids.     

Deletion of the entire cytochrome P450 CYP2D6 gene as a cause of impaired drug metabolism in poor metabolizers of the debrisoquine/sparteine polymorphism.

The debrisoquine/sparteine polymorphism is associated with a clinically important genetic deficiency of oxidative drug metabolism. From 5% to 10% of Caucasians designated as poor metabolizers (PMs) of the debrisoquine/sparteine polymorphism have a severely impaired capacity to metabolize more than 25 therapeutically used drugs. The impaired drug metabolism in PMs is due to the absence of cytochrome P450IID6 protein. The gene controlling the P450IID6 protein, CYP2D6, is located on the long arm of chromosome 22. A pseudogene CYP2D8P and a related gene CYP2D7 are located upstream from CYP2D6. This gene locus is highly polymorphic. After digestion of genomic DNA with XbaI endonuclease, restriction fragments of 11.5 kb and 44 kb represent mutant alleles of the cytochrome CYP2D6 gene locus associated with the PM phenotype. In order to elucidate the molecular mechanism of the mutant allele reflected by the XbaI 11.5-kb fragment, a genomic library was constructed from leukocyte DNA of one individual homozygous for this fragment and screened with the human IID6 cDNA. The CYP2D genes were isolated and characterized by restriction mapping and partial sequencing. We demonstrate that the mutant 11.5-kb allele results from a deletion involving the entire functional CYP2D6 gene. This result provides an explanation for the total absence of P450IID6 protein in the liver of these PMs.     

Muscle force arises by actin filament rotation and torque in the Z-filaments

Cytochrome P450 partial sequences were isolated by PCR using genomic DNA from two hymenopteran insects of agronomical importance, Trichogramma cacoeciae, a parasitoid wasp, and Apis mellifera, the honeybee. Four new P450 genes were identified: one honeybee gene belongs to the CYP4 family and was named CYP4G11; the three other genes were from Trichogramma and belong to the CYP4 family (CYP4G12) and to a novel family, the CYP48 one (CYP48A1 and CYP48A2). The four genes contain a short intron (72-95 bp) at the same position as already described for other insect species. The two genes CYP48A1 and CYP48A2 have a supernumary intron (57-71 bp) upstream the first one. Only the two CYP4 genes were constitutively transcribed, at a high level for CYP4G12 and at a low level for CYP4G11. No expression was observed for CYP48A1 and CYP48A2.     

Genomic clustering of cyanogenic glucoside biosynthetic genes aids their identification in Lotus japonicus and suggests the repeated evolution of this chemical defence pathway

Cyanogenic glucosides are amino acid-derived defence compounds found in a large number of vascular plants. Their hydrolysis by specific β-glucosidases following tissue damage results in the release of hydrogen cyanide. The cyanogenesis deficient1 (cyd1) mutant of Lotus japonicus carries a partial deletion of the CYP79D3 gene, which encodes a cytochrome P450 enzyme that is responsible for the first step in cyanogenic glucoside biosynthesis. The genomic region surrounding CYP79D3 contains genes encoding the CYP736A2 protein and the UDP-glycosyltransferase UGT85K3. In combination with CYP79D3, these genes encode the enzymes that constitute the entire pathway for cyanogenic glucoside biosynthesis. The biosynthetic genes for cyanogenic glucoside biosynthesis are also co-localized in cassava (Manihot esculenta) and sorghum (Sorghum bicolor), but the three gene clusters show no other similarities. Although the individual enzymes encoded by the biosynthetic genes in these three plant species are related, they are not necessarily orthologous. The independent evolution of cyanogenic glucoside biosynthesis in several higher plant lineages by the repeated recruitment of members from similar gene families, such as the CYP79s, is a likely scenario.     

Metabolic activation of the pneumotoxin, 3-methylindole, by vaccinia-expressed cytochrome P450s

Twelve human cytochrome P450s and one mouse P450 were produced in HepG2 cells using vaccinia virus cDNA expression and analyzed for their ability to bioactivate the pneumotoxin, 3-methylindole (3MI), to an electrophilic metabolite(s) which alkylated cellular macromolecules. Cell lysates containing CYP2C8, CYP3A4, CYP2A6 and CYP2F1 metabolized 3MI to an intermediate(s) that became covalently bound to lysate material. A control lysate produced from cells which had been infected with a wild-type vaccinia virus was not able to bioactivate 3MI. The mouse 1A2 enzyme metabolized 3MI at a rate of 75.4 pmol/mg protein/minute, while the rate of metabolism in the lysate containing the human 1A2 P450 enzyme was not different from that in the control lysate. Therefore, the catalytic capabilities of orthologous P450 enzymes to activate 3MI cannot be extrapolated among different species. These results indicate that human P450s are capable of bioactivating 3MI to a metabolite which binds to cellular macromolecules suggesting that this compound may be toxic to humans.     

Multispecies comparative analysis of a mammalian-specific genomic domain encoding secretory proteins

The mammalian-specific casein gene cluster comprises 3 or 4 evolutionarily related genes and 1 physically linked gene with a functional association. To gain a better understanding of the mechanisms regulating the entire casein cluster at the genomic level we initiated a multispecies comparative sequence analysis. Despite the high level of divergence at the coding level, these studies have identified uncharacterized family members within two species and the presence at orthologous positions of previously uncharacterized genes. Also the previous suggestion that the histatin/statherin gene family, located in this region, was primate specific was ruled out. All 11 genes identified in this region appear to encode secretory proteins. Conservation of a number of noncoding regions was observed; one coincides with an element previously suggested to be important for beta-casein gene expression in human and cow. The conserved regions might have biological importance for the regulation of genes in this genomic "neighborhood."     

The cytochrome P450 gene superfamily in Drosophila melanogaster: annotation, intron-exon organization and phylogeny

The cytochrome P450 gene superfamily is represented by 90 sequences in the Drosophila melanogaster genome. Of these 90 P450 sequences, 83 code for apparently functional genes whereas seven are apparent pseudogenes. More than half of the genes belong to only two families, CYP4 and CYP6. The CYP6 family is insect specific whereas the CYP4 family includes sequences from vertebrates. There are eight genes coding for mitochondrial P450s as deduced from their homology to CYP12A1 from the house fly. The genetic map of the distribution of D. melanogaster P450 genes shows (a) the absence of P450 genes on the chromosome 4 and Y, (b) more than half of the P450 genes are found on chromosome 2, and (c) the largest cluster contains nine genes. Sequence alignments were used to draw phylogenetic trees and to analyze the intron-exon organization of each functional P450 gene. Only five P450 genes are intronless. We found 57 unique intron positions, of which 23 were phase zero, 19 were phase one and 15 were phase two. There was a relatively good correlation between intron conservation and phylogenetic relationship between members of the P450 subfamilies. Although the function of many P450 proteins from vertebrates, fungi, plants and bacteria is known, only a single P450 from D. melanogaster, CYP6A2, has been functionally characterized. Gene organization appears to be a useful tool in the study of the regulation, the physiological role and the function of these P450s.     

Expression of a cytochrome P450 gene family in maize

Maize seedlings, like seedlings of many other plants, are rich in cytochrome P450 (P450) enzyme activity. Four P450 genes (CYPzm1–4), isolated from a seedling-specific cDNA library, are characterised by a transient and seedling-specific expression pattern. The maximum steady state mRNA levels are reached at 3 days in root and at 7 days in shoot tissue, respectively. All four genes belong to one gene family and are closely related to the CYP71 family of plant P450 genes, which includes the enzymes of the ripening avocado fruit (CYP71A1) and eggplant hypocotyls (CYP71A2, A3, A4). The expression of these related P450 genes in monocot and dicot plants indicates that these enzymes play a significant role in plants; however, the in vivo enzyme functions are unknown. The divergence of the four members of the maize gene family is sufficiently high to account for different substrate and/or reaction specificity. Although the general expression pattern of the four genes is identical, the maximum steady-state mRNA levels vary in different maize lines. In situ hybridisation reveals the highest mRNA levels in the coleoptile, the first developed leaflets, the ground tissue of the nodular complex, and in the cortex and pith of the region of cell division in the root. The mapping of the maize CYPzm genes shows that, as in animals, P450 genes of the same family can be clustered. The presence of the CYPzm gene cluster in maize argues for generation of distinct plant P450 gene families by gene duplication.     

Comparison of P450s from human and fugu: 420 million years of vertebrate P450 evolution

The fugu (pufferfish) genome has been sequenced, and a second genome assembly was released 17 May 2002. Exhaustive searches were made to identify all P450 genes and pseudogenes from the earlier release of 26 October 2001. P450 genes assembled as completely as possible from these data were used to do additional searches of the newer assembly and all P450 genes and pseudogenes in the available fugu sequence data have been identified, compared to human P450s, and assigned names. There are 54 P450 genes in fugu and 1 nearly intact pseudogene (CYP3A50P). CYP1A is missing much of its N-terminal half; however, 45 P450 genes are completely assembled. Eight others are lacking only one or two exons or less. CYP2X4 is known only from an EST. This may be a 55th P450 gene if it represents an accurate sequence. In addition to 2X4, there are 16 other pseudogene fragments or small pieces of P450 genes. At the P450 family level, 17 of 18 mammalian families are found in fugu. CYP39 is the only CYP family missing and it is not seen in any other fish sequence data either. The CYP2 family shows the largest degree of divergence. In the CYP2 family, only CYP2R1 and CYP2U1 are conserved as recognizable subfamilies across species. Intron-exon boundaries are largely preserved across 420 million years of evolution.