Identification of a multi-component berberine 11-hydroxylase from Burkholderia sp. strain CJ1

ABSTRACT

Berberine (BBR) is a protoberberine alkaloid extracted from plants such as Coptis japonica (Ranunculaceae). In a previous report, we demonstrated the existence of a 11-hydroxylation pathway employed by BBR-utilizing bacteria for metabolism of BBR. In the present study, we report the identification of the genes brhA, brhB, and brhC as encoding a multicomponent BBR 11-hydroxylase in Burkholderia sp. strain CJ1. BrhA is belonging to the Rieske non-heme iron oxygenase (RO) family, a class of enzymes known to catalyze the first step in bacterial aromatic-ring hydroxylation. We further demonstrate that BrhA activity requires BrhB (ferredoxin reductase) and BrhC (ferredoxin) as electron transport chain components. A BLAST search revealed that BrhA exhibits 38% and 33% sequence identity to dicamba O-demethylase (DdmC; AY786443) and chloroacetanilide herbicides N-dealkylase (CndA; KJ461679), respectively. To our knowledge, this work represents the first report of a bacterial oxygenase catalyzing the metabolism of a polycyclic aromatic-ring alkaloid.

Abbreviations: BBR: berberine; D-BBR: demethyleneberberine; H-BBR: 11-hydroxyberberine; HD-BBR: 11-hydroxydemethyleneberberine; HDBA: 2-hydroxy-3,4-dimethoxybenzeneacetic acid; PAL: palmatine; H-PAL: 11-hydroxypalmatine; BRU: berberrubine; Fd: ferredoxin; FdR: ferredoxin reductase; ETC: electron transport chain     

Bioavailability Study of Berberine and the Enhancing Effects of TPGS on Intestinal Absorption in Rats

Berberine chloride (BBR) is a natural isoquinoline alkaloid extracted from medicinal herbs. It has been reported that the intestinal absorption of BBR is very low. In this study, the absolute bioavailability of BBR was studied, and the enhancing effects of d-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) on intestinal absorption were investigated in rats. BBR injection was administrated via the femoral vein at a dose of 1.0 mg kg⁻¹ in intravenous group, and BBR oral formulations were administrated by oral gavage at a dose of 100 mg kg⁻¹ in BBR control (control) group and BBR-TPGS (test) group, respectively. The result showed that BBR had a very low absolute bioavailability of 0.68%, and TPGS could enhance intestinal absorption of BBR significantly. TPGS at a concentration of 2.5% could improve peak concentration (C _max) and area under the curve (AUC_0–36) of BBR by 2.9 and 1.9 times, respectively. The absorption enhancing ability of TPGS may be due to its ability to affect the biological activity of P-glycoprotein and thereby reduce the excretion of absorbed BBR into the intestinal lumen. This study indicated that absolute bioavailability of BBR was 0.68% in rats, and TPGS was a good absorption enhancer capable of enhancing intestinal absorption of BBR significantly.     

Therapeutic effect of oxyberberine on obese non-alcoholic fatty liver disease rats

BackgroundBerberine (BBR) has been widely used to treat non-alcoholic fatty liver disease (NAFLD). The metabolites of BBR were believed to contribute significantly to its pharmacological effects. Oxyberberine (OBB), a gut microbiota-mediated oxidative metabolite of BBR, has been firstly identified in our recent work.PurposeHere, we aimed to comparatively investigate the anti-NAFLD properties of OBB and BBR.MethodsThe anti-NAFLD effect was evaluated in high-fat diet-induced obese NAFLD rats with biochemical/ELISA tests and histological staining. The related gene and protein expressions were detected by qRT-PCR and Western blotting respectively. Molecular docking and dynamic simulation were also performed to provide further insight.ResultsResults indicated OBB remarkably and dose-dependently attenuated the clinical manifestations of NAFLD, which (100 mg/kg) achieved similar therapeutic effect to metformin (300 mg/kg) and was superior to BBR of the same dose. OBB significantly inhibited aberrant phosphorylation of IRS-1 and up-regulated the downstream protein expression and phosphorylation (PI3K, p-Akt/Akt and p-GSK-3β/GSK-3β) to improve hepatic insulin signal transduction. Meanwhile, OBB treatment remarkably alleviated inflammation via down-regulating the mRNA expression of MCP-1, Cd68, Nos2, Cd11c, while enhancing Arg1 mRNA expression in white adipose tissue. Moreover, OBB exhibited closer affinity with AMPK in silicon and superior hyperphosphorylation of AMPK in vivo, leading to increased ACC mRNA expression in liver and UCP-1 protein expression in adipose tissue.ConclusionTaken together, compared with BBR, OBB was more capable of maintaining lipid homeostasis between liver and WAT via attenuating hepatic insulin pathway and adipocyte inflammation, which was associated with its property of superior AMPK activator.     

黄连素对小鼠心肌梗死后心室重构的作用研究

目的:探讨黄连素(Berberine,BBR)在小鼠心肌梗死(myocardial infarction,MI)后心室重构中的作用,并比较BBR预处理(BBR pre-treatment,preBBR)和BBR后处理(BBR post-treatment,postBBR)给药的效果。方法:将60只C57BL/6小鼠随机分为4组,分别为假手术组、单纯MI对照组、MI+preBBR组及MI+postBBR组,每组15只。MI模型采用前降支结扎法制备。MI+preBBR组在MI模型制备前2周开始用BBR(100 mg·kg~(-1)·d~(-1))每天灌胃,持续至MI后28 d;MI+postBBR组在MI模型制备后4 h开始用BBR(100 mg·kg~(-1)·d~(-1))每天灌胃,持续至MI后28 d。记录实验期间小鼠生存情况。MI后28天采用小动物超声测定左心室收缩功能;取心脏组织,测定心脏大小和重量;ELISA方法测定血浆BNP水平;Masson染色评价心肌纤维化程度。结果:与单纯MI对照组相比,MI+preBBR组及MI+postBBR组小鼠生存率提高、心脏收缩功能增强、心脏变小、心脏重量减轻、血浆BNP水平降低、心肌纤维化明显改善。其中,MI+preBBR组上述指标的改善程度优于MI+postBBR组。结论:BBR可抑制MI后心室重构,且BBR预处理效果优于后处理。     

Cytochrome P450 activities in pure and co-cultured rat hepatocytes. Effects of model inducers

Summary The stability and inducibility of several P450 activities (namely, P450 1A1, 2A1, 2B1/2, 2C11, and 3A1) were studied in rat hepatocytes co-cultured with the MS epithelial cell line derived from monkey kidney. The results revealed that these monooxygenase activities were systematically higher in co-cultures than in conventional hepatocyte cultures. Pure cultures showed a rapid loss of monooxygenase activities, which were undetectable after 5 days. In contrast, all isozymes assayed were measurable in co-cultured hepatocytes on Day 7 (about 15 to 40% of the initial activities of Day 0 of culture). The beneficial effects of the co-culture system seemed to be more selective for certain cytochrome P450 isoforms, with P450 1A1 and 3A1 being the best stabilized isozymes after 1 wk. A clear response to inducers was observed in co-cultures, each isozyme showing a different induction pattern. 3-Methylcholanthrene produced a strong increase in P450 1A1 (7-ethoxyresorufin O-deethylase) activity and a low increase in P450 2A1 (testosterone 7α-hydroxylation), whereas no changes were observed in the other activities. Phenobarbital treatment resulted in increases in P450 2B1/2 (7-pentoxyresorufin O-depentylase and 16α- and 16β-hydroxylation of testosterone) activities, while minor effects were observed on P450 3A1 (testosterone 6β-hydroxylation) activity. Dexamethasone markedly increased P450 3A1 (testosterone 6β- and 15β-hydroxylation) activity and, to a lesser extent, P450 2B1/2 (16β-hydroxylation).     

Biotransformation of (20S)-20-hydroxymethylpregna-1,4-dien-3-one by four filamentous fungi

Microbial transformation of (20S)-20-hydroxymethylpregna-1,4-dien-3-one (1) by four filamentous fungi, Cunninghamella elegans, Macrophomina phaseolina, Rhizopus stolonifer, and Gibberella fujikuroi, afforded nine new, and two known metabolites 2–12. The structures of these metabolites were characterized through detailed spectroscopic analysis. These metabolites were obtained as a result of biohydroxylation of 1 at C-6β, -7β, -11α, -14α, -15β, -16β, and -17α positions, except metabolite 2 which contain an O-acetyl group at C-22. These fungal strains demonstrated to be efficient biocatalysts for 11α-hydroxylation. Compound 1, and its metabolites were evaluated for the first time for their cytotoxicity against the HeLa cancer cell lines, and some interesting results were obtained.     

The renoprotective effects of berberine via the EP4-Gαs-cAMP signaling pathway in different stages of diabetes in rats

Abstract

Aims: To investigate the renoprotective roles of berberine (BBR) in different stages of diabetic nephropathy (DN) in streptozotocin (STZ)-induced diabetic rats fed a high-sugar and high-fat diet. Methods: Diabetes was induced in mice by intraperitoneal injection of STZ, and the mice were then randomly divided into groups: normal, diabetes, high-sugar and high-fat and BBR (high, median and low dose) groups. The body weight (BW), kidney weight to body weight (KW/BW), blood urea nitrogen, urine total protein to urine creatinine ratio and serum creatinine were measured on different weeks throughout the study. The protein levels of E prostanoid receptor 4 (EP4), Gαs and content of cAMP in the kidney were, respectively, detected by western blot analysis and RIA analysis. Results: In the DN rats, there was remarkable renal damage. BBR restored renal functional parameters, suppressed alterations in histological and ultrastructural changes in the kidney tissues and increased EP4, Gαs and cAMP levels compared with those of the DN model group. In addition, BBR has different therapeutic effects during the different stages of the development of DN, and it works best in the sixth week. Conclusion: These studies demonstrate, for the first time, that BBR exerts renoprotective effects in different stages of DN via EP4- Gαs- AC-cAMP signaling pathway in STZ-induced DN rats fed a high-sugar and high-fat diet.     

Pedobacter aquae sp. nov., a multi-drug resistant bacterium isolated from fresh water

A novel bacterial strain designated CJ43^T was isolated from fresh water located in Gangwon-do, South Korea, displaying multi-drug resistance. The isolate was Gram-stain-negative, aerobic, orange-pigmented, and rod-shaped. Strain CJ43^T grew optimally at 30 °C and pH 7 on R2A agar in the absence of NaCl. Phylogenetic analyses based on 16S rRNA gene sequences revealed that strain CJ43^T belonged to the genus Pedobacter in the family Sphingobacteriaceae and was most closely related to Pedobacter puniceum HX-22-1 ^T and P. glucosidilyticus 1-2 ^T (98.3 and 98.1% sequence similarity). The genome size of strain CJ43^T was 3.9 Mb in a single contig with DNA G?+?C content of 34.9%. The genome included 3144 predicted protein-coding genes, as well as 55 tRNA, 9 rRNA and 3 ncRNA genes. The genome also contained 128 putative antibiotic resistance genes, reflecting its phenotypes. The average nucleotide identity values between strain CJ43^T and two closely related strains P. puniceum HX-22-1 ^T and P. glucosidilyticus 1-2 ^T were 91.0 and 88.7%, respectively. In silico digital DNA-DNA hybridization results between strain CJ43^T and the related strains were 42.8 and 38.6%, respectively. The major fatty acids of strain CJ43^T were iso-C_15:0, iso-C_17:0 3-OH, and summed feature 3 (C_16:1 ω6c and/or C_16:1 ω7c). Strain CJ43^T contained phosphatidylethanolamine as the major polar lipid and menaquinone-7 as the sole respiratory quinone. Based on the polyphasic taxonomy data, strain CJ43^T represents a novel species of the genus Pedobacter, for which the name Pedobacter aquae sp. nov. is proposed with the type strain CJ43^T (=?KACC 21350 ^T?=?JCM 33709 ^T).

     

The genome of Polaromonas naphthalenivorans strain CJ2, isolated from coal tar-contaminated sediment, reveals physiological and metabolic versatility and evolution through extensive horizontal gene transfer

We analysed the genome of the aromatic hydrocarbon‐degrading, facultatively chemolithotrophic betaproteobacterium, Polaromonas naphthalenivorans strain CJ2. Recent work has increasingly shown that Polaromonas species are prevalent in a variety of pristine oligotrophic environments, as well as polluted habitats. Besides a circular chromosome of 4.4 Mb, strain CJ2 carries eight plasmids ranging from 353 to 6.4 kb in size. Overall, the genome is predicted to encode 4929 proteins. Comparisons of DNA sequences at the individual gene, gene cluster and whole‐genome scales revealed strong trends in shared heredity between strain CJ2 and other members of the Comamonadaceae and Burkholderiaceae. blastp analyses of protein coding sequences across strain CJ2's genome showed that genetic commonalities with other betaproteobacteria diminished significantly in strain CJ2's plasmids compared with the chromosome, especially for the smallest ones. Broad trends in nucleotide characteristics (GC content, GC skew, Karlin signature difference) showed at least six anomalous regions in the chromosome, indicating alteration of genome architecture via horizontal gene transfer. Detailed analysis of one of these anomalous regions (96 kb in size, containing the nag‐like naphthalene catabolic operon) indicates that the fragment's insertion site was within a putative MiaB‐like tRNA‐modifying enzyme coding sequence. The mosaic nature of strain CJ2's genome was further emphasized by the presence of 309 mobile genetic elements scattered throughout the genome, including 131 predicted transposase genes, 178 phage‐related genes, and representatives of 12 families of insertion elements. A total of three different terminal oxidase genes were found (putative cytochrome aa₃‐type oxidase, cytochrome cbb₃‐type oxidase and cytochrome bd‐type quinol oxidase), suggesting adaptation by strain CJ2 to variable aerobic and microaerobic conditions. Sequence‐suggested abilities of strain CJ2 to carry out nitrogen fixation and grow on the aromatic compounds, biphenyl and benzoate, were experimentally verified. These new phenotypes and genotypes set the stage for gaining additional insights into the physiology and biochemistry contributing to strain CJ2's fitness in its native habitat, contaminated sediment.     

Differential effect of Shenmai injection, a herbal preparation, on the cytochrome P450 3A-mediated 1′-hydroxylation and 4-hydroxylation of midazolam

Shenmai injection (SMI), one of the most popular herbal preparations, is widely used for the treatment of coronary atherosclerotic cardiopathy and viral myocarditis. The purpose of this study was to investigate the effect of Shenmai injection (SMI) on the CYP3A-mediated metabolism of midazolam (MDZ). The present study demonstrated that SMI could significantly inhibit MDZ 4-hydroxylation but activate its 1′-hydroxylation in human liver microsomes (HLMs), rat liver microsomes (RLM) and recombinant human CYP3A4 and CYP3A5. The opposing effect of SMI was characterized by the kinetic change of increasing V_max/K_m for MDZ 1′-hydroxylation and decreasing V_max/K_m for MDZ 4-hydroxylation in HLM and RLM. The presence ofSMI enhanced the inhibition of ketoconazole on MDZ 4-hydroxylation but weakened or reversed its inhibition on MDZ 1′-hydroxylation in HLM. After single or multiple pretreatment with SMI, the ratios of AUC_{4-OH MDZ}/AUC_MDZ in rats were significantly decreased, while the ratios of AUC_{1′-OH MDZ}/AUC_MDZ were increased. Among the major components in SMI, total ginsenoside (TG), ophiopogon total saponins (OTS), ophiopogon total flavone (OTF), ginsenoside Rd, ophiopogonin D and ophiopogonone A exhibited significant inhibition on both 4-hydroxylation and 1′-hydroxylation of MDZ in HLM and RLM, while no activation on MDZ metabolism was observed in the presence of these major constituents alone or together. To further explore the responsible components, 3 mL of SMI was loaded on a solid phase extraction (SPE) C₁₈ cartridge and then separated by different concentrations of methanol. The fractions eluted with 60% and 90% methanol both showed significant activation on MDZ 1′-hydroxylation in HLM, but the fraction eluted with 30% methanol had no such effect. The results indicated that the activation of SMI on MDZ 1′-hydroxylation might be mainly resulted from the lipid-soluble components in SMI.     

Studies on phytoremediation potentiality of Typhonium flagelliforme for the degradation of Brilliant Blue R

In vitro culture plants of Typhonium flagelliforme were found to decolorize a variety of dyes, including Malachite Green, Red HE 8B, Methyl Orange, Reactive Red 2, Direct Red 5B (DR5B), Red HE 7B, Golden Yellow HER, Patent Blue, and Brilliant Blue R (BBR), to varying extents within 4 days. The enzymatic analysis of plant roots of aseptically raised plantlets performed before and after degradation of the dye BBR by these plantlets showed a significant induction in the activities of peroxidase, laccase, tyrosinase, and 2,6-dichlorophenol-indophenol reductase, which indicated the involvement of these enzymes in the metabolism of the dye. Comparative study of the enzyme status of the plants Typhonium flagelliforme and Blumea malcolmii during the degradation of DR5B and BBR showed marked variations in the enzyme profile with respect to the use of different sources of the enzyme. Phytoremediation of BBR using Typhonium flagelliforme was confirmed with high performance liquid chromatography and Fourier transform infrared spectroscopy analysis performed before and after the degradation of the dye. One of the products of the metabolism of the dye was identified as 4-(4-ethylimino-cyclohexa-2,5-dienylidinemethyl)-phenylamine with the aid of gas chromatography–mass spectroscopy (GC–MS) analysis. Significant decrease in the American Dye Manufacturer’s Institute, biological oxygen demand, and chemical oxygen demand values of synthetic mixture of textile dyes and industrial effluent confirmed the decolorization and detoxification. Phytotoxicity studies also revealed the nontoxic nature of the metabolites of BBR.     

Role of nitrogen fixation in the autecology of Polaromonas naphthalenivorans in contaminated sediments

Polaromonas naphthalenivorans strain CJ2 is a Gram‐negative betaproteobacterium that was identified, using stable isotope probing in 2003, as a dominant in situ degrader of naphthalene in coal tar‐contaminated sediments. The sequenced genome of strain CJ2 revealed several genes conferring nitrogen fixation within a 65.6 kb region of strain CJ2's chromosome that is absent in the genome of its closest sequenced relative Polaromonas sp. strain JS666. Laboratory growth and nitrogenase assays verified that these genes are functional, providing an alternative source of nitrogen in N‐free media when using naphthalene or pyruvate as carbon sources. Knowing this, we investigated if nitrogen‐fixation activity could be detected in microcosms containing sediments from the field site where strain CJ2 was isolated. Inducing nitrogen limitation with the addition of glucose or naphthalene stimulated nitrogenase activity in amended sediments, as detected using the acetylene reduction assay. With the use of fluorescence microscopy, we screened the microcosm sediments for the presence of active strain CJ2 cells using a dual‐labelling approach. When we examined the carbon‐amended microcosm sediments stained with both a strain CJ2‐specific fluorescent in situ hybridization probe and a polyclonal fluorescently tagged antibody, we were able to detect dual‐labelled active cells. In contrast, in sediments that received no carbon addition (showing no nitrogenase activity), no dual‐labelled cells were detected. Furthermore, the naphthalene amendment enhanced the proportion of active strain CJ2 cells in the sediment relative to a glucose amendment. Field experiments performed in sediments where strain CJ2 was isolated showed nitrogenase activity in response to dosing with naphthalene. Dual‐label fluorescence staining of these sediments showed a fivefold increase in active strain CJ2 in the sediments dosed with naphthalene over those dosed with deionized water. These experiments show that nitrogen fixation may play an important role in naphthalene biodegradation by strain CJ2 and contribute to its ecological success.     

Inhibition of human cytochromes P450 2A6 and 2A13 by flavonoids,acetylenic thiophenes and sesquiterpene lactones from Pluchea indica and Vernonia cinerea

The human liver cytochrome P450 (CYP) 2A6 and the respiratory CYP2A13 enzymes play role in nicotine metabolism and activation of tobacco-specific nitrosamine carcinogens. Inhibition of both enzymes could offer a strategy for smoking abstinence and decreased risks of respiratory diseases and lung cancer. In this study, activity-guided isolation identified four flavonoids 1–4 (apigenin, luteolin, chrysoeriol, quercetin) from Vernonia cinerea and Pluchea indica, four hirsutinolide-type sesquiterpene lactones 5–8 from V. cinerea, and acetylenic thiophenes 9–11 from P. indica that inhibited CYP2A6- and CYP2A13-mediated coumarin 7-hydroxylation. Flavonoids were most effective in inhibition against CYP2A6 and CYP2A13, followed by thiophenes, and hirsutinolides. Hirsutinolides and thiophenes exhibited mechanism-based inhibition and in irreversible mode against both enzymes. The inactivation kinetic K_I values of hirsutinolides against CYP2A6 and CYP2A13 were 5.32–15.4 and 0.92–8.67 µM, respectively, while those of thiophenes were 0.11–1.01 and 0.67–0.97 µM, respectively.     

Cloning and expression of a trehalose synthase from Pseudomonas stutzeri CJ38 in Escherichia coli for the production of trehalose

A novel strain was isolated, Pseudomonas stutzeri CJ38, that enabled direct transformation of maltose to trehalose. In comparison with others reported to date, CJ38 provided a novel trehalose synthase (TSase) without any byproduct, including glucose. Activity analysis, using either maltose or trehalose as a substrate, showed a reversible reaction. There was also no detectable activity of related enzymes with liquid starch and maltooligosaccharides as substrates. Using a malPQ-negative host and MacConkey medium, the TSase gene was cloned in Escherichia coli from CJ38. The resulting sequence contained an open reading frame consisted of 689 amino acids with a calculated molecular mass of 76 kDa. A search for related sequences in various gene and protein data banks revealed a novel family of enzymes that was predicted putatively as a glycosidase or TSase family, with no biochemical evidence. The recombinant enzyme exhibited a high activity toward the substrate maltose, about 50-fold higher than the parent strain and resulted in a high conversion yield (72%) at a relatively high substrate concentration (20%). These results provided the possibility that the strain was effectively used as a potential biocatalyst for the production of trehalose from maltose in a one-step reaction.     

反刍动物产志贺毒素大肠杆菌的分离鉴定和致病潜力分析

产志贺毒素大肠杆菌（Shiga toxin-producing Escherichia coli,STEC）是重要的食源性病原,而STEC往往以正常菌群的形式存在于牛羊等反刍动物肠道。[目的] 本研究对牛羊粪便样品中的STEC分离和鉴定并对分离株进行致病潜力分析。从江苏、云南和河北等地共分离到羊源STEC菌株11株,牛源STEC菌株1株,另新疆农业大学佟盼盼组馈赠牛源菌株10株。[方法] 通过细菌选择培养及特异性基因stx1和stx2的检测进行分离鉴定;并通过Vero细胞毒性试验、溶血活性试验和毒力因子的检测分析STEC分离株的致病潜力。[结果] 分离到羊源分离株11株,分离率17.5%（11/63）;分离得到牛源分离株1株,分离率0.7%（1/134）;11株羊源分离株中有5株对Vero细胞具有强的毒性,3株有溶血活性;11株牛源分离株中有5株对Vero细胞具有强的毒性,3株有溶血活性。11株羊源STEC分离株eae基因携带率为63.6%（7/11）,而11株牛源STEC分离株eae基因携带率仅为9.0%（1/11）。[结论] 结果表明羊源STEC菌株的分离率和致病潜力高于牛源菌株,所以,除牛外,羊作为STEC菌株宿主也应该得到更多的重视。     

Biotransformation of 3β-hydroxy-5-en-steroids by Mucor silvaticus

Abstract

The biotransformation of four 3β-hydroxy-5-en-steroids with varying substituents at C-16 or/and C-17 by Mucor silvaticus was investigated. The characterization of the metabolites was performed by IR, MS, ¹H NMR, ¹³C NMR, and 2-D NMR. All the examined substrates were transformed, mainly by 7α-hydroxylation. Studies carried out with M. silvaticus demonstrated the versatility of this organism in introducing hydroxyl groups at the 7α-, 9α-, 11α-, and 14α-positions in 3-ol-5-ene steroids. The relationships between the substrate structures and hydroxylated positions are also discussed.     

Immunization with a dominant-negative recombinant Herpes Simplex Virus (HSV) type 1 protects against HSV-2 genital disease in guinea pigs

Background  

CJ9-gD is a novel dominant-negative recombinant herpes simplex virus type 1 (HSV-1) that is completely replication-defective, cannot establish detectable latent infection in vivo, and expresses high levels of the major HSV-1 antigen glycoprotein D immediately following infection. In the present study, CJ9-gD was evaluated as a vaccine against HSV-2 genital infection in guinea pigs.     

New Genes on Human Chromosome 7: Bioinformation Analysis of a Cluster of Genes from the POLR2J Family

Four independent genes encoding various variants of the hRPB11 subunit of Homo sapiens RNA polymerase II were revealed in human chromosome 7. Three genes (POLR2 J1, POLR2 J2, and POLR2 J3) form a cluster of total length of 214 530 bp in the genetic locus 7q22.1 on the long arm of chromosome 7 (contig NT_007933). The fourth gene (POLR2 J4, 31 040 bp) was localized in the cytogenetic locus 7p13 of the short arm of chromosome 7 (contig NT_007819). An analysis enabled us to refine dissimilar experimental data on the mapping of the hRPB11 subunit gene on chromosome 7. In particular, the presence of three sites of its localization according to data on hybridization with fluorescent-labeled probes (the FISH method) was explained. It was established that, upon the expression of the four human POLR2 J genes, at least 14 types of mature mRNAs encoding somewhat differing hRPB11 isoforms can be synthesized. Eleven of these mRNAs were revealed (as full-length copies or clearly identifiable fragments) in the available databases of expressed sequence tags and cDNAs. The most probable scheme of origination of the multiple genes of the POLR2 J family as a result of three consecutive segmented duplications increasing in size was proposed and substantiated. On the basis of the scheme, some assumptions on the pathways of evolution of separate human genes and the mechanisms of generation of protein diversity in higher eukaryotes were made.