Characterization of multiple <Emphasis Type="Italic">CYP9A</Emphasis> genes in the silkworm, <Emphasis Type="Italic">Bombyx mori</Emphasis>

Based on the advances in the silkworm genome project, a new genome-wide analysis of cytochrome P450 genes was performed, focusing mainly on gene duplication. All four CYP9A subfamily members from the silkworm, Bombyx mori, were cloned by RT-PCR and designated CYP9A19–CYP9A22 by the P450 Nomenclature Committee. They each contain an open reading frame of 1,593 bp in length and encode a putative polypeptide of 531 amino acids. Both nucleic acid and amino acid sequences share very high identities with one another. The typical motifs of insect cytochrome P450, including the heme-binding region, helix-C, helix-I, helix-K, and PERF, show high sequence conservation among the multiple proteins. Alignment with their cDNA sequences revealed that these paralogues share identical gene structures, each comprising ten exons and nine introns of variable sizes. The locations of their introns (all nine introns follow the GT–AG rule) are absolutely conserved. CYP9A19, CYP9A20, and CYP9A21 form a tandem cluster on chromosome 17, whereas CYP9A22 is separated from the cluster by four tandem alcohol-dehydrogenase-like genes. Their phylogenetic relationships and structural comparisons indicated that these paralogues arose as the results of gene duplication events. RT-PCR detected their mRNAs in different “first line of defense” tissues, as well as in several other organs, suggesting diverse functions. Tissue-selective expression also indicates their functional divergence. The identified CYP9A genes have not yet been found outside the Lepidoptera, and are probably unique to the Lepidoptera. They show high sequence and structural similarities to each other, indicating that the Lepidoptera-specific P450s may be of functional importance. This analysis constitutes the first report of the clustering, spatial organization, and functional divergence of P450 in the silkworm.     

Analysis of fragment homology among DNA sequences from cytochrome P450 families 4 and 6

Cytochrome P450s comprise a diverse superfamily of proteins that often share as little as 12% amino acid identity. Accordingly, the identification of novel gene families, subfamilies and alleles has been based primarily on ‘benchmark’ levels of global amino acid identity and, more recently, phylogenetic relatedness has been used to resolve ambiguous relationships. However, PCR-based cloning strategies have resulted in a large increase in the number of short DNA sequences, particularly among insects. Many of these fragments remain unnamed and even their gene family membership remains unknown due to the uncertainty as to whether these fragments accurately reflect the levels of sequence identity or patterns of evolutionary divergence exhibited by groups of full-length P450 sequences. As a result, the nature of P450 diversity among insects remains obscure. In this paper, P450 sequences belonging to the two major gene families, that is, families CYP4 and CYP6, are analyzed by comparing segments to full-length sequences. A parameter called the Segmental Divergence Index is used to characterize segments of P450s with respect to the degree to which they mirror the divergence of full-length sequences. The reliability of these fragments in phylogenetic analyses is also tested. The results of these analyses support the use of some commonly amplified P450 gene fragments for naming new P450 genes and for studies of P450 diversity and evolution. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

Genome-wide analysis of cytochrome P450 monooxygenase genes in the silkworm, Bombyx mori

Based on the advances in the silkworm genome project, a new genome-wide analysis of cytochrome P450 genes was performed. A total of 84 CYP-related sequences were identified and could be classified into 26 families and 47 subfamilies according to standard nomenclature. Seventy eight of the eighty four genes appear to be functional and six are probable pseudogenes. The distribution of Bombyx mori P450s in the genome shows that most of them are tandem arranged on chromosomes, only 34 genes are present as singletons, with 8 clusters including 3 or more than 3 genes. Sequence alignments were used to reconstruct phylogenetic trees and to analyze the intron-exon organizations of the functional genes. The conserved intron positioning agrees perfectly with their common grouping on the tree. The presence of three extremely ancient introns which are conserved across different clans indicates that a few introns are still highly conserved after they have undergone extensive evolutionary changes of B. mori P450 duplication and divergence. Comparison of the P450s from B. mori to the P450s from Drosophila melanogaster shows that the expansion is not uniform across the gene families. Remarkably, two mitochondrial families, the B. mori CYP333 and D. melanogaster Cyp12, formed two orthologous groups in the phylogenetic tree. All CYP333s can be proposed to be related to xenobiotic metabolism in accordance with the D. melanogaster Cyp12s. The characterization and evolutionary analysis of P450s from B. mori in the current study provide useful information for understanding the characteristics and diversity of P450s from B. mori and the baseline for functional analyses of individual P450s in this model Lepidopteran insect.     

淡色库蚊细胞色素P450基因研究

采用一对昆虫细胞色素P450简并引物,以反转录-聚合酶链反应从淡色库蚊对溴氰菊酯敏感品系和抗性品系成虫RNA扩增到约485 bp和510 bp两个片段,将这两个片段与PinPoint^TMXa-1 T质粒重组,然后克隆至大肠杆菌JM109菌株,筛选获得68个阳性克隆;其中24个阳性克隆测序后与GenBank资料对照,显示为细胞色素P450新序列;分子系统学研究显示,24个新基因（等位基因）分别属CYP4家族CYP4C、CYP4D、CYP4H和CYP4J等4个亚家族,其已由细胞色素P450命名委员会命名和GenBank登录上网;其中CYP4C23可能是一个假基因,CYP4H13具有一段58个碱基长度的内含子,CYP4J4V1在近3′端具有一个终止密码子TAG.     

Intron-exon organization and phylogeny in a large superfamily, the paralogous cytochrome P450 genes of Arabidopsis thaliana

The cytochrome P450 gene superfamily is represented by 80 genes in animal genomes and perhaps more than 300 genes in plant genomes. We analyzed about half of all Arabidopsis P450 genes, a very large dataset of truly paralogous genes. Sequence alignments were used to draw phylogenetic trees, and this information was compared with the intron-exon organization of each P450 gene. We found 60 unique intron positions, of which 37 were phase 0 introns. Our results confirm the polyphyletic origin of plant P450 genes. One group of these genes, the A-type P450s, are plant specific and characterized by a simple organization, with one highly conserved intron. Closely related A-type P450 genes are often clustered in the genome with as many as a dozen genes (e.g., of the CYP71 subfamily) on a short stretch of chromosome. The other P450 genes (non-A-type) form several distinct clades and are characterized by numerous introns. One such clade contains the two CYP51 genes, which are thought to encode obtusifoliol 14a demethylase. The two CYP51 genes have a single intron that is not shared with CYP51 genes from vertebrates or fungi, or with any other Arabidopsis P450 gene. Only a few of the Arabidopsis P450 genes are intronless (e.g., the CYP710A and CYP96A subfamilies). There was a relatively good correlation between intron conservation and phylogenetic relationships between members of the P450 subfamilies. Gene organization appears to be a useful tool in establishing the evolutionary relatedness of P450 genes, which may help in predictions of P450 function.     

热带假丝酵母1230细胞色素P450基因CYPA14与CYPA16的克隆和表达

细胞色素P450(CYP)是一种单加氧酶,在热带假丝酵母(Candidatropicalis)ω-氧化过程中发挥关键作用。通过对来源不同的P450基因进行同源性分析,首先克隆到热带假丝酵母1230中P450基因的部分序列,再利用基因组步行法克隆其未知序列,结果分别获得了两个P450同工酶基因CYPA14和CYPA16的完整序列。经PCR方法证实,二者在染色体上的位置相邻,其读码框分别编码522和540个氨基酸残基的肽链。经NCBIBLAST搜索比较后发现,二者与热带假丝酵母ATCC20336中的P450成员CYP52A14和CYP52A16分别编码的序列几乎完全一致,与热带假丝酵母ATCC750中的P450成员CYP52A2和CYP52A1也具有较高的相似性。同时,对经诱变后的几株二元酸生产菌株的CYPA14与CYPA16也进行了克隆和序列比较,发现部分序列中的个别氨基酸残基发生了突变。CYPA14和CYPA16均在酿酒酵母中获得了有效表达,其中CYPA16的P450表达含量高于CYPA14,后者有部分表达产物发生了变性。     

Isolation and sequence analysis of P450 genes from a pyrethroid resistant colony of the major malaria vector Anopheles funestus.

Pyrethroid resistance has been demonstrated in populations of Anopheles funestus from South Africa and southern Mozambique. Resistance is associated with elevated P450 monooxygenase enzymes. In this study, degenerate primers based on conserved regions of Anopheles gambiae P450 CYP4, 6 and 9 families were used to amplify genomic and cDNA templates from A. funestus. A total of 12 CYP4, 12 CYP6 and 7 CYP9 partial genes have been isolated and sequenced. BLAST results revealed that A. funestus P450s generally have a high sequence identity to A. gambiae with above 75% identity at the amino acid level. The exception is CYP9J14. The A. gambiae P450 showing highest identity to CYP9J14 exhibits only 55% identity suggesting that CYP9J14 may have arisen from a recent duplication event. Molecular phylogenetic analysis based on amino acid sequences also supported this hypothesis. Intron positions, but not size, were highly conserved between the two species. The high level of orthology that exists in the P450 gene families of these two species may facilitate the prediction of individual P450 protein function.     

豌豆蚜基因组P450基因家族的分析

细胞色素P450在各种内源和外源物质代谢中起着非常重要的作用。利用豌豆蚜Acyrthosiphon pisum基因组mRNA和氨基酸数据库研究P450基因功能的进化规律。通过生物信息学方法对豌豆蚜全基因组P450进行分析, 结果显示: 在豌豆蚜基因组中发现69个P450基因, 它们分别属于13个P450家族和18个亚家族, 是一个典型的多基因家族。进一步将这些基因与豌豆蚜ESTs数据库进行了比对分析, 其中39个候选基因有EST证据, 证明了这些P450基因在转录水平的真实性。以氨基酸相似度大于60%为标准对豌豆蚜基因组中P450基因进行分组, 69个P450基因中, 除18个基因序列因差异太大, 不能被归入任何一组, 其余51个可归入10个组, 其中8个组(包含47条序列)适合于正选择和基因转换分析。正选择和基因转换分析结果表明: 仅有1个组(含9个基因)显著受到正选择压力作用, 正选择概率大于95%的氨基酸位点分别是20T和27N, 20T位于底物识别位点SRS1, 27N位于D. helix; 有3个组(包含8个基因）显示显著的基因转换事件。 参与基因转换的基因均为CYP4家族成员, 分别是CYP4C, CYP4G和CYP4V亚家族。 参与基因转换的成员之间的蛋白相似度较高（70％~95％）, 且XM_001944991与XM_001951794同存在于SCAFFOLD12542上, XM_001945510与XM_001944057同存在于SCAFFOLD7010上。这可能暗示豌豆蚜P450基因通过基因复制, 然后通过基因转换使P450获得新的功能, 以适应多变的生存环境。此外, 鉴定出20个不同的基序, 其中有5条基序在90％以上的基因中出现。      

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.     

A new family of cytochrome P450 genes (CYP41) from the cattle tick, Boophilus microplus.

We isolated and sequenced a cytochrome P450 (CYP) gene that is sufficiently different from other CYP genes that a new CYP family, CYP41 was created. CYP41 encodes a protein of 518 residues and is most similar to genes from the family CYP3; it is 36% identical to CYP3A2 and 34% identical to CYP3A28. We hypothesise that CYP41 encodes an enzyme that metabolizes xenobiotic compounds i.e. compounds that are foreign to the cattle tick. The phylogenetic position of CYP41 could not be resolved because of the high level of sequence divergence at both the nucleotide and amino acid levels.     

MOLECULAR CLONING AND SEQUENCE ANALYSIS OF THREE NEW FULL‐LENGTH cDNAs OF CYTOCHROME P450 (CYP6N3v1‐v3)*FROM AEDES ALBOPICTUS**

Abstract The three new full‐length cDNA sequences including the complete 5′‐and 3′‐ untranslated regions (UTR) coding for cytochrome P450s from Aedes albopictus have been obtained. The P450 proteins deduced from the nucleotide sequences shared 58.6% ‐ 62.4% amino acid identity with CYP6N1 and CYP6N2 from Anopheles gambiae, and 99% with each other. The three new complete sequences have been submitted and named as CYP6N3v1, CYP6N3v2 and CYP6N3v3 by the P450 Nomenclature Committee. The original cDNAs were obtained by rapid amplification of cDNA ends (RACE) approach with several pairs of gene specific primers based on the cDNA fragment previously obtained from deltamethrin‐resistant strain of Ae. albopictus. Further analysis showed that the three new sequences are present in both resistant strain and susceptible strain and might be effectively translated. In addition, the 5′‐ and 3′‐UTRs were compared between the CYP6N3vl‐v3 and other known insect P450s. The multiplicity of trans‐lational control of insect P450 genes was discussed.     

Isolation, characterization, and chromosomal mapping of mouse P450 17 alpha-hydroxylase/C17-20 lyase.

Cytochrome P450 17 alpha-hydroxylase/C17-20 lyase (P45017 alpha) catalyzes the conversion of C-21 steroids to C-19 steroids in gonads. A full-length mouse cDNA encoding P450 17 alpha was isolated from a mouse Leydig cell library and characterized by restriction mapping and sequencing. The predicted amino acid sequence has 83% homology to rat, 66% homology to human, and 62% homology to bovine P45017 alpha amino acid sequences. The protein is 507 amino acids in length, which is 1 amino acid shorter than the human protein and 2 amino acids shorter than the bovine protein. The structural gene encoding P450 17 alpha (Cyp17) was localized utilizing an interspecific testcross to mouse chromosome 19, distal to Got-1. Another cytochrome P450, P4502c (Cyp2c), also is located at the distal end of chromosome 19. CYP17, CYP2c, and GOT1 have been mapped to human chromosome 10, with CYP2C and GOT1 mapped to the distal region, q24.3 and q25.3, respectively. The data in the present study indicate conserved syntenic loci on mouse chromosome 19 and human chromosome 10 and predict that the structural gene encoding P45017 alpha will be found distal to GOT1 on human chromosome 10.     

Characterization of the cytochrome P-450IID subfamily in bovine liver. Nucleotide sequences and microheterogeneity.

To elucidate the molecular mechanisms underlying drug detoxification, the structures of the members of the microsomal cytochrome P-450IID subfamily were analyzed by isolating, mapping and sequencing cytochrome P-450IID (CYP2D) cDNA clones from bovine liver. The screening was performed under nonstringent conditions so that most of the P-450IID subfamily members could be obtained. 114 of the 147 positive clones were classified into four groups on the basis of their restriction-enzyme maps. The maps of the four groups were highly similar, however, the clones of one group contained an insertion of approximately 500 bp in the coding region. Analysis of partial nucleotide sequences of several representative clones from each group showed that the bovine P-450IID subfamily in liver consisted of several, not many, highly similar members, differing by less than 7% in their nucleotide sequences. The location of the insertion found in the minor group corresponded to intron 7 and the GT/AG rule was found at the exon/intron boundary, suggesting that intron 7 was retained in this group. The complete nucleotide sequences of two clones from the major group were examined to determine the structures of the P-450IID subfamily in bovine liver. A full-length cDNA clone (1615 bp) and a partial cDNA clone (1538 bp) contained open reading frames encoding 500 and 487 amino acid residues, respectively. The partial clone lacked the nucleotide sequence corresponding to the first 13 N-terminal amino acid residues. The deduced amino acid sequences of the two clones were 98% similar, and 80% and 68% similar to those from human CYP2D6 and rat CYP2D1, respectively. Comparisons of the amino acid sequences of the P-450IID subfamily members showed the highly conserved C-terminal region of their molecules and the high similarity between the members in one species, especially in cattle and man.