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Results for “Nested Genes”

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PubMed1996

Expression of cytokine genes and presence of enteroviral genomic RNA in endomyocardial biopsy tissues of myocarditis and dilated cardiomyopathy.

Viral infection, especially by enteroviruses, has been considered to be the most common cause of myocarditis, which may progress to dilated cardiomyopathy (DCM). Although the mechanism of progression remains uncertain, a cytokine-associated injury of myocytes has been proposed. Using reverse transcriptase polymerase chain reaction (RT-PCR), we examined the expression of interleukin 1 beta (IL-1 beta), IL-6, IL-8 and tumour necrosis factor alpha (TNF-alpha) and the presence of enteroviral genomic RNA in endomyocardial biopsy tissues obtained from patients with myocarditis and DCM. We examined endomyocardial biopsy tissues obtained from 6 patients with myocarditis, 21 with DCM and 15 with non-infectious cardiac diseases as controls. In patients with myocarditis, endomyocardial biopsy was performed twice at an interval of 1 month to 8 years after the onset of myocarditis. We used RT-PCR to detect IL-1 beta, IL-6, IL-8 and TNF-alpha genes expression and nested RT-PCR (nRT-PCR) to detect enteroviral genomic RNA. IL-1 beta, IL-6, IL-8 and TNF-alpha genes were expressed in 100% (6/6) and enteroviral genomic RNA in 67% (4/6) of myocarditis patients at the first biopsy. At the second biopsy, IL-1 beta, IL-6, IL-8 and TNF-alpha genes were expressed in none, 50% (3/6), 67% (4/6) and 67% (4/6), respectively, and enteroviral genomic RNA in 67% (4/6). Four patients with myocarditis, in whom IL-8 and TNF-alpha genes and enteroviral genomic RNA were detected, progressed to DCM at the second biopsy. IL-1 beta, IL-6, IL-8 and TNF-alpha genes were expressed in none, 24% (5/21), 38% (8/21), 57% (12/21) of DCM patients, respectively. Enteroviral genomic RNA was detected in 43% (9/21) of DCM. Neither cytokine expression nor enteroviral genomic RNA were detected in the controls. the high incidence of cytokines, especially IL-6, IL-8 and TNF-alpha, expression in myocarditis and DCM, which might be induced by enteroviral infection, suggests that cytokines play an important role in myocytic damage leading to DCM.

Adult↗

PubMedDate not supplied

The relationship between the degree of thrombocytopenia and infection with Ehrlichia canis in an endemic area.

Ehrlichia canis is the causative agent of canine monocytic ehrlichiosis. In order to evaluate platelet counts as a screening test for E. canis in an endemic area, 217 whole blood samples from dogs were divided into three groups: 71 non-thrombocytopenic samples (group A, platelet counts greater than 200 000/mL) and 146 thrombocytopenic samples (less than 200 000/mL). The thrombocytopenic group was further divided into 62 with platelet counts between 100 000-200 000/mL (Group B) and 84 samples with less than 100 000 platelets/mL (Group C). All samples were examined for the presence of a segment of the Ehrlichia canis 16S rRNA gene using a nested polymerase chain reaction. Sixty-seven of the 217 samples (30.9%) were positive for the presence of the E. canis 16S rRNA gene; 53 (63.1%) of the group C samples and 13 (21%) of group B. Only one (1.4%) of the non-thrombocytopenic samples (Group A) was positive. These data support the concept that platelet counts may be a good screening test for canine monocytic ehrlichiosis, and that the magnitude of thrombocytopenia may increase the reliability of diagnosis.

Animals↗

PubMed2005

Application of nested polymerase chain reaction (nPCR) using MPB 64 gene primers to detect Mycobacterium tuberculosis DNA in clinical specimens from extrapulmonary tuberculosis patients.

BACKGROUND & OBJECTIVE: The conventional culture technique for diagnosis of extrapulmonary tuberculosis is time consuming. In order to find a sensitive and rapid technique nested polymerase chain reaction (nPCR) targeting the conserved MPB 64 gene of Mycobacterium tuberculosis was evaluated for detection of M. tuberculosis DNA directly from clinical specimens of extrapulmonary origin. METHODS: A total of 400 clinical specimens from clinically suspected cases of extrapulmonary tuberculosis and 30 control specimens of nontuberculous aetiology were processed by smear and culture and by nPCR technique for detection of M. tuberculosis. The specimens were divided into 3 groups, (group I--280 specimens [104 peritoneal fluid (PF), 120 cerebrospinal fluid (CSF), 44 lymph node biopsies 3 pericardial fluid and 9 other biopsy specimens], group II--120 aqueous humour (AH) from idiopathic granulomatous uveitis cases, and group III--30 control specimens (10 CSF and 20AH). RESULTS: The conventional culture was positive only in 16 of 400 specimens. The overall positivity of nPCR was 35.2 per cent (141/400). Among the 280 specimens from extrapulmonary lesions (group I), 15 were bacteriologically positive, while 115 of 265 bacteriologically negative specimens (43.4%) were positive by nPCR. All the 30 control specimens were negative by nPCR. INTERPRETATION & CONCLUSION: The nPCR using MPB64 gene primers might be a rapid and reliable diagnostic technique for detection of M. tuberculosis genome in clinically suspected extrapulmonary tuberculosis specimens, as compared to the conventional techniques.

Aqueous Humor↗

PubMed2005

[Detection of bcl-2/IgH gene rearrangement in diffuse large B cell lymphoma by hemi-nested PCR].

OBJECTIVES: To explore a sensitive and specific method for detection of bcl-2/IgH gene rearrangement in diffuse large B cell lymphoma (DLBCL), and verify the credibility of the established method. METHODS: bcl-2/IgH hemi-nested PCR primers were designed using the professional primer design software. Fifty-two samples of pathologically diagnosed DLBCL and 10 fresh tonsil tissues were amplified using hemi-nested touch down-PCR to detect bcl-2/IgH gene rearrangement. The PCR products were cloned and sequenced. RESULTS: bcl-2/IgH gene rearrangement was detected in 6 of 52 DLBCL samples and 2 of 10 fresh tonsil tissues using one-way method. By using the hemi-nested PCR for the second round amplification, 5 of DLBCL were positive, but all of the fresh tonsil tissues were negative. The positive PCR products were sequenced and analyzed on the Internet, 3 of 8 cases obtained by one-way method were false positive, 5 positive cases amplified using hemi-nested PCR were all bcl-2/IgH gene rearrangement. PCR products of 3 false positive cases were homologous to BAC331191 and LLNLR-245D11 in human chromosome 19 and RP11-498P10 in chromosome 1. CONCLUSION: There are false positive results using common primers for detecting bcl-2/IgH gene rearrangement. The mechanism may be that highly homologous sequences to human genome exist in commonly used primers. The specificity of the diagnosis could be improved by hemi-nested PCR using the combination of primers we designed and the traditional ones.

Gene Rearrangement, B-Lymphocyte, Heavy Chain↗

PubMed2002

Cardiomyogenic differentiation in cardiac myxoma expressing lineage-specific transcription factors.

We investigated five cases of cardiac myxoma and one case of cardiac undifferentiated sarcoma by light and electron microscopy, in situ hybridization, immunohistochemical staining, and reverse transcriptase-polymerase chain reaction for cardiomyocyte-specific transcription factors, Nkx2.5/Csx, GATA-4, MEF2, and eHAND. Conventional light microscopy revealed that cardiac myxoma and sarcoma cells presented variable cellular arrangements and different histological characteristics. Ultrastructurally, some of the myxoma cells exhibited endothelium-like or immature mesenchymal cell differentiation. Immunohistochemistry for Nkx2.5/Csx, GATA-4, and eHAND was slightly to intensely positive in all myxoma cases. MEF2 immunoreactivity was observed in all cases including the case of sarcoma, thus suggesting myogenic differentiation of myxoma or sarcoma cells. In situ hybridization for Nkx2.5/Csx also revealed that all myxoma cells, but not sarcoma cells, expressed mRNA of the cardiac homeobox gene, Nkx2.5/Csx. Furthermore, nested reverse transcriptase-polymerase chain reaction from formalin-fixed, paraffin-embedded tissue was performed and demonstrated that the Nkx2.5/Csx and eHAND gene product to be detected in all cases, and in three of six cases, respectively. In conclusion, cardiac myxoma cells were found to express various amounts of cardiomyocyte-specific transcription factor gene products at the mRNA and protein levels, thus suggesting cardiomyogenic differentiation. These results support the concept that cardiac myxoma might arise from mesenchymal cardiomyocyte progenitor cells.

Aged↗

PubMed2000

Primary cutaneous B-cell lymphoma and Borrelia burgdorferi infection in patients from the Highlands of Scotland.

Although a link between primary cutaneous B-cell lymphoma (PCBCL) and Borrelia burgdorferi infection has long been suspected, previous studies have not demonstrated a significant association. The authors looked for evidence of B. burgdorferi in 20 cases of PCBCL from the Scottish Highlands, an area with endemic Lyme disease, and compared their findings with those in 40 control patients (20 undergoing wide reexcision at sites of malignant melanoma and 20 biopsies of inflammatory dermatoses). All studies were performed on formalin-fixed, paraffin-embedded tissues. The cases of PCBCL were classified according to criteria described by the European Organization for Research and Treatment of Cancer Cutaneous Lymphoma Project Group using a combination of morphology, immunohistochemistry, and seminested polymerase chain reaction (PCR) for immunoglobulin heavy chain gene rearrangement. A nested PCR was performed on deoxyribonucleic acid (DNA) extracts from the lymphoma and control cases using primers to a unique conserved region of the B. burgdorferi flagellin gene. B. burgdorferi-specific DNA was detected in seven of 20 lymphoma cases (five of 12 marginal zone lymphomas, one of five primary cutaneous follicle center cell lymphomas, one of three diffuse, large B-cell lymphomas of the leg) and in one melanoma reexcision patient of 40 control subjects. The relationship between B. burgdorferi and PCBCL was significant when compared with the control groups separately (p <0.05) or in combination (p <0.01). These results provide strong evidence to support the concept of B. burgdorferi-driven lymphomagenesis in the skin.

Adult↗

PubMed2002

Evaluation of two nested PCR assays for detection of Histoplasma capsulatum DNA in human tissue.

In order to evaluate the diagnostic relevance of two nested PCR assays for diagnosis of histoplasmosis in clinical specimens, 100 paraffin-embedded biopsy specimens were examined. Upon microscopy of tissue, 50 biopsy specimens were histoplasma positive and 50 were negative. Due to destruction by formalin fixation, successful extraction of amplifiable human DNA was limited to 29 and 33 samples, respectively. A product of the Histoplasma capsulatum nested PCR assay targeting the gene encoding the unique fungal 100-kDa-like protein was detected in 20 histopathologically positive biopsy specimens but in none of the microscopically negative samples. Sequencing revealed that all 20 products of 210 bp were identical to the sequence of H. capsulatum in the GenBank database. In contrast, the nested PCR assay targeting the fungal 18S rRNA genes amplified products in 26 histopathologically positive but also in 18 microscopically negative biopsy specimens. However, sequencing revealed that only 20 of these 44 PCR products (231 bp) were identical to the sequence of H. capsulatum. The remaining 24 sequences were homologous to those of several Euascomycetes. These PCR products were detected only in tissues possibly colonized by nonpathogenic fungi, possibly causing these nonspecific amplifications. The detection limit of both H. capsulatum nested PCR assays was 1 to 5 fungal cells per sample. The two assays were similarly sensitive in identifying H. capsulatum. In this preliminary study, the novel 100-kDa-like-protein gene nested PCR revealed a specificity of 100% without requiring sequencing, which was necessary for identification of the 18S ribosomal DNA nested PCR products in order to avoid a high rate of false-positive results.

Cloning, Molecular↗

PubMed2000

A case of orbital lymphoproliferative lesion diagnosed as malignant lymphoma after recurring 11 years later

Background: Most primary lymphoproliferative lesions in the ocular adnexa, including the eyelid, conjunctiva, and orbit, are diagnosed as low-grade malignant lymphomas. Recurrence and dissemination of these tumors are rare in Japan. The long-term prognosis for this disorder still remains to be clarified.Case and Method: A 53-year-old woman was first referred to us for right orbital tumor in 1986. After subtotal resection of the tumor, the patient received no additional treatment. She visited us in 1997 with the complaint of bilateral orbital tumor. Biopsied specimens were examined histologically using hematoxylin-eosin and immunohistological staining. Southern blot hybridization was used to detect immunoglobulin gene rearrangement. The paraffin-embedded specimen obtained in 1986 was also examined for immunoglobulin gene rearrangement using nested polymerase chain reaction technique. Findings: The specimens from 1997 and 1986 were both diagnosed as lymphoid type of inflammatory pseudotumor, based on polyclonal B cell immunohistological staining. Immunoglobulin gene rearrangement was present in both specimens.Conclusions: The orbital tumor resected in 1986 was a low-grade malignant lymphoma which disseminated systematically 11 years later. This case shows a long-term course of orbital lymphoproliferative lesion with positive immunoglobulin gene rearrangement. It also shows the importance of follow-up over 10 years in the case of low-grade malignant lymphoma of the ocular adnexa.

Journal Article↗

PubMed1999

[A case of orbital lymphoproliferative lesion diagnosed as malignant lymphoma after recurring 11 years later].

BACKGROUND: Most primary lymphoproliferative lesions in the ocular adnexa, including the eyelid, conjunctiva, and orbit, are diagnosed as low-grade malignant lymphomas. Recurrence and dissemination of these tumors are rare in Japan. The long-term prognosis for this disorder still remains to be clarified. CASE AND METHOD: A 53-year-old woman was first referred to us for right orbital tumor in 1986. After subtotal resection of the tumor, the patient received no additional treatment. She visited us in 1997 with the complaint of bilateral orbital tumor. Biopsied specimens were examined histologically using hematoxylin-eosine and immunohistological staining. Southern blot hybridization was used to detect immunoglobulin gene rearrangement. The paraffin-embedded specimen obtained in 1986 was also examined for immunoglobulin gene rearrangement using nested polymerase chain reaction technique. FINDINGS: The specimens from 1997 and 1986 were both diagnosed as lymphoid type of inflammatory pseudotumor, based on polyclonal B cell immunohistological staining. Immunoglobulin gene rearrangement was present in both specimens. CONCLUSION: The orbital tumor resected in 1986 was a low-grade malignant lymphoma which disseminated systemically 11 years later. This case shows a long-term course of orbital lymphoproliferative lesion with positive immunoglobulin gene rearrangement. It also shows the importance of follow-up over 10 years in the case of low-grade malignant lymphoma of the ocular adnexa.

Diagnosis, Differential↗

PubMed1997

The NTN2L gene encoding a novel human netrin maps to the autosomal dominant polycystic kidney disease region on chromosome 16p13.3.

The netrins define a family of chemotropic factors that have been shown to play a central role in axon guidance. We identified two exon traps encoding netrin-like sequences during the assembly of a transcriptional map for the genomic interval surrounding the polycystic kidney disease type 1 and tuberous sclerosis type 2 genes. We describe the characterization of a novel human netrin-2-like gene, designated NTN2L, and its transcript. The genomic interval containing the NTN2L gene was sequenced, and the coding region was predicted based on computer analysis. The structure of the NTN2L gene has been confirmed utilizing nested RT-PCR. The NTN2L gene is predicted to encode a 580-amino-acid protein having homology to the chicken and Drosophila netrins and to Caenorhabditis elegans UNC-6. The NTN2L gene has a restricted pattern of expression; its transcript is undetectable by Northern analysis in all tissues examined, but can be recovered from spinal cord RNA by RT-PCR. This report represents the first description and characterization of a human netrin.

Amino Acid Sequence↗

PubMed2005

Evaluation of candidate gene effects for beef backfat via Bayesian model selection.

Candidate gene approaches provide tools for exploring and localizing causative genes affecting quantitative traits and the underlying variation may be better understood by determining the relative magnitudes of effects of their polymorphisms. Diacyglycerol O-acyltransferase 1 (DGAT1), fatty acid binding protein (heart) 3 (FABP3), growth hormone 1 (GH1), leptin (LEP) and thyroglobulin (TG) have been previously identified as genes contributing to genetic control of subcutaneous fat thickness (SFT) in beef cattle. In the present research, Bayesian model selection was used to evaluate effects of these five candidate genes by comparing competing non-nested models and treating candidate gene effects as either random or fixed. The analyses were implemented in SAS to simplify the programming and computation. Phenotypic data were gathered from a F(2) population of Wagyu x Limousin cattle. The five candidate genes had significant but varied effects on SFT in this population. Bayesian model selection identified the DGAT1 model as the one with the greatest model probability, whether candidate gene effects were considered random or fixed, and DGAT1 had the greatest additive effect on SFT. The SAS codes developed in the study are freely available and can be downloaded at: http://www.ansci.wsu.edu/programs/.

Adiposity↗

PubMed2005

I-ApeKI [corrected]: a novel intron-encoded LAGLIDADG homing endonuclease from the archaeon, Aeropyrum pernix K1.

Over 50 introns have been reported in archaeal rRNA genes (rDNAs), a subset of which nests putative homing endonuclease (HEase) genes. Here, we report the identification and characterization of a novel archaeal LAGLIDADG-type HEase, I-ApeKI [corrected], encoded by the ApeK1.S908 intron within the 16S rDNA of Aeropyrum pernix K1. I-ApeKI [corrected] consists of 222 amino acids and harbors two LAGLIDADG-like sequences. It recognizes the 20 bp non-palindromic sequence 5'-GCAAGGCTGAAAC downward arrowTTAAAGG and cleaves target DNA to produce protruding tetranucleotide 3' ends. Either Mn2+ or Co2+ can be substituted for Mg2+ as a cofactor in the cleavage reaction. Of the 20 bases within the minimal recognition site, 7 are essential for cleavage and are located at positions proximal to the cleavage sites.

Aeropyrum↗

PubMed1991

Promoter of the adenovirus polypeptide IX gene: similarity to E1B and inactivation by substitution of the simian virus 40 TATA element.

The promoter of the adenovirus polypeptide IX (pIX) gene consists of an SP1 binding site and a TATA box and is remarkably similar to the promoter of the E1B gene in which it is nested. Plasmid constructs containing the pIX gene with deletions in the SP1 or TATA sites were defective in pIX mRNA production in transient expression assays. These results were confirmed with analogous virus constructs. An oligonucleotide containing sequences within the pIX promoter region spanning the SP1 and TATA sites but not including the sequences downstream of the TATA box is sufficient to direct mRNA synthesis at +90 nucleotides within the pIX gene. While the simian virus 40 (SV40) early promoter is capable of directing pIX mRNA synthesis from the SV40 cap sites, substitution of the pIX TATA box with the SV40 TATA box results in barely detectable levels of pIX mRNA. These results will be discussed with respect to exchangeability of promoter elements and the possible role of the viral E1B 21-kDa protein in potentiating or stabilizing transcription factor TFIID binding to the pIX TATA element.

Adenovirus Early Proteins↗

PubMed2003

[Gene expression analysis in liver tissue at a single cell level by nested polymerase chain reaction and laser microdissection].

OBJECTIVE: To investigate the measurements of gene expressing at a single hepatocyte level. METHODS: Individual hepatocyte was isolated from cryostat tissue section using laser microdissection technique. To detect the mRNA expressed by single hepatocyte, RNA was extracted, reversely transcribed to cDNA and amplified by nested polymerase chain reaction (PCR). RESULTS: Single cell was microdissected from cryostat tissue using an ultraviolet laser micromanipulator. The RNA could be extracted from the isolated cell(s), and the RT-PCR production could be observed after electrophoresis, whose quantitation was compatible with the number of cells. CONCLUSION: Combining laser microdissection and nested RT-PCR can monitor gene expression at a single cell level in vivo.

Dissection↗

PubMed2005

A gene truncation strategy generating N- and C-terminal deletion variants of proteins for functional studies: mapping of the Sec1p binding domain in yeast Mso1p by a Mu in vitro transposition-based approach.

Bacteriophage Mu in vitro transposition constitutes a versatile tool in molecular biology, with applications ranging from engineering of single genes or proteins to modification of genome segments or entire genomes. A new strategy was devised on the basis of Mu transposition that via a few manipulation steps simultaneously generates a nested set of gene constructions encoding deletion variants of proteins. C-terminal deletions are produced using a mini-Mu transposon that carries translation stop signals close to each transposon end. Similarly, N-terminal deletions are generated using a transposon with appropriate restriction sites, which allows deletion of the 5'-distal part of the gene. As a proof of principle, we produced a set of plasmid constructions encoding both C- and N-terminally truncated variants of yeast Mso1p and mapped its Sec1p-interacting region. The most important amino acids for the interaction in Mso1p are located between residues T46 and N78, with some weaker interactions possibly within the region E79-N105. This general-purpose gene truncation strategy is highly efficient and produces, in a single reaction series, a comprehensive repertoire of gene constructions encoding protein deletion variants, valuable in many types of functional studies. Importantly, the methodology is applicable to any protein-encoding gene cloned in an appropriate vector.

Bacteriophage mu↗

PubMed2000

Precursor genes of future pandemic influenza viruses are perpetuated in ducks nesting in Siberia.

Influenza A viruses of different subtypes were isolated from fecal samples of ducks in their nesting areas in Siberia in summer from 1996 to 1998. Phylogenetic analysis of the NP genes of the isolates in Siberia and those in Hokkaido, Japan on their flyway of migration from Siberia to the south in autumn revealed that they belong to the Eurasian lineage of avian influenza viruses. It is noted that the genes of the isolates in Siberia are closely related to those of H5N1 influenza virus strains isolated from chickens and humans in Hong Kong in 1997 as well as to those of isolates from domestic birds in southern China. The results indicate that influenza viruses perpetuated in ducks nesting in Siberia should have contributed genes in the emergence of the H5N1 virus in Hong Kong. Vaccine prepared from avirulent A/duck/Hokkaido/4/96 (H5N3) influenza virus was potent enough to protect mice from challenge with lethal dose of the pathogenic H5N1 virus [19]. Intensive surveillance study of aquatic birds especially in Siberia is, therefore, stressed to provide information on the future pandemic influenza virus strains and for vaccine preparation.

Animals↗

PubMed1998

Identification of a glucocorticoid response element in the rat beta2-adrenergic receptor gene.

Regulation of beta2-adrenergic receptor (beta2AR) levels by glucocorticoids is a physiologically important mechanism for altering beta2AR responsiveness. Glucocorticoids increase beta2AR density by increasing the rate of beta2AR gene transcription, but the cis-elements involved have not been well characterized. We now show that one of six potential glucocorticoid response elements (GREs) in the 5'-flanking region of the rat beta2AR gene is necessary for glucocorticoid-dependent stimulation of receptor gene expression. Using a nested set of deletion fragments of the rat beta2AR gene 5'-flanking region fused to a luciferase reporter gene, glucocorticoid-dependent induction of reporter gene expression in HepG2 cells was localized to a region between positions -643 and -152, relative to the transcription initiation site. In electrophoretic mobility shift assays, a double-stranded oligonucleotide incorporating a near-consensus GRE from this region (positions -379 to -365) formed complexes with the human recombinant glucocorticoid receptor, as well as with nuclear protein from dexamethasone-treated HepG2 cells. Mutation of a single base within this GRE sequence greatly diminished interaction of the mutated oligonucleotide with the human recombinant glucocorticoid receptor. The functional activity of the GRE was characterized using a luciferase reporter construct driven by a minimal thymidine kinase promoter. In HepG2 cells transfected with constructs containing the GRE, dexamethasone increased reporter gene expression approximately 3-fold, whereas a dexamethasone effect was not observed with constructs lacking the GRE. Taken together, these findings show that a GRE located at positions -379 to -365 in the 5'-flanking region of the rat beta2AR gene mediates glucocorticoid stimulation of beta2AR gene transcription.

Animals↗

PubMed2001

Complete nucleotide sequence and organization of the atrazine catabolic plasmid pADP-1 from Pseudomonas sp. strain ADP.

The complete 108,845-nucleotide sequence of catabolic plasmid pADP-1 from Pseudomonas sp. strain ADP was determined. Plasmid pADP-1 was previously shown to encode AtzA, AtzB, and AtzC, which catalyze the sequential hydrolytic removal of s-triazine ring substituents from the herbicide atrazine to yield cyanuric acid. Computational analyses indicated that pADP-1 encodes 104 putative open reading frames (ORFs), which are predicted to function in catabolism, transposition, and plasmid maintenance, transfer, and replication. Regions encoding transfer and replication functions of pADP-1 had 80 to 100% amino acid sequence identity to pR751, an IncPbeta plasmid previously isolated from Enterobacter aerogenes. pADP-1 was shown to contain a functional mercury resistance operon with 99% identity to Tn5053. Complete copies of transposases with 99% amino acid sequence identity to TnpA from IS1071 and TnpA from Pseudomonas pseudoalcaligenes were identified and flank each of the atzA, atzB, and atzC genes, forming structures resembling nested catabolic transposons. Functional analyses identified three new catabolic genes, atzD, atzE, and atzF, which participate in atrazine catabolism. Crude extracts from Escherichia coli expressing AtzD hydrolyzed cyanuric acid to biuret. AtzD showed 58% amino acid sequence identity to TrzD, a cyanuric acid amidohydrolase, from Pseudomonas sp. strain NRRLB-12227. Two other genes encoding the further catabolism of cyanuric acid, atzE and atzF, reside in a contiguous cluster adjacent to a potential LysR-type transcriptional regulator. E. coli strains bearing atzE and atzF were shown to encode a biuret hydrolase and allophanate hydrolase, respectively. atzDEF are cotranscribed. AtzE and AtzF are members of a common amidase protein family. These data reveal the complete structure of a catabolic plasmid and show that the atrazine catabolic genes are dispersed on three disparate regions of the plasmid. These results begin to provide insight into how plasmids are structured, and thus evolve, to encode the catabolism of compounds recently added to the biosphere.

Amino Acid Sequence↗