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PubMed2005

Diversity, localization, and physiological properties of filamentous microbes belonging to Chloroflexi subphylum I in mesophilic and thermophilic methanogenic sludge granules.

We previously reported that the thermophilic filamentous anaerobe Anaerolinea thermophila, which is the first cultured representative of subphylum I of the bacterial phylum Chloroflexi, not only was one of the predominant constituents of thermophilic sludge granules but also was a causative agent of filamentous sludge bulking in a thermophilic (55 degrees C) upflow anaerobic sludge blanket (UASB) reactor in which high-strength organic wastewater was treated (Y. Sekiguchi, H. Takahashi, Y. Kamagata, A. Ohashi, and H. Harada, Appl. Environ. Microbiol. 67:5740-5749, 2001). To further elucidate the ecology and function of Anaerolinea-type filamentous microbes in UASB sludge granules, we surveyed the diversity, distribution, and physiological properties of Chloroflexi subphylum I microbes residing in UASB granules. Five different types of mesophilic and thermophilic UASB sludge were used to analyze the Chloroflexi subphylum I populations. 16S rRNA gene cloning-based analyses using a 16S rRNA gene-targeted Chloroflexi-specific PCR primer set revealed that all clonal sequences were affiliated with the Chloroflexi subphylum I group and that a number of different phylotypes were present in each clone library, suggesting the ubiquity and vast genetic diversity of these populations in UASB sludge granules. Subsequent fluorescence in situ hybridization (FISH) of the three different types of mesophilic sludge granules using a Chloroflexi-specific probe suggested that all probe-reactive cells had a filamentous morphology and were widely distributed within the sludge granules. The FISH observations also indicated that the Chloroflexi subphylum I bacteria were not always the predominant populations within mesophilic sludge granules, in contrast to thermophilic sludge granules. We isolated two mesophilic strains and one thermophilic strain belonging to the Chloroflexi subphylum I group. The physiological properties of these isolates suggested that these populations may contribute to the degradation of carbohydrates and other cellular components, such as amino acids, in the bioreactors.

Chloroflexi↗
PubMed2006

Fine-scale differences between Accumulibacter-like bacteria in enhanced biological phosphorus removal activated sludge.

A lab-scale sequencing batch reactor (SBR) and six full-scale wastewater treatment plants (WWTPs) performing enhanced biological phosphorus removal (EBPR) were surveyed. The abundance of Accumulibacter-related organisms in the full-scale plants was investigated using fluorescent in situ hybridization. Accumulibacter-related organisms were present in all of the full-scale EBPR plants, at levels ranging from 9% to 24% of total cells. The high percentage of Accumulibacter-related organisms seemed to be associated with configurations which minimize the nitrate recycling to the anaerobic zone and low influent BOD:TP ratios. PCR-based clone libraries were constructed from the community 16S rRNA gene plus the internally transcribed spacer region amplified from the SBR and five of the full-scale WWTPs. Comparative sequence analysis was carried out using Accumulibacter-related clones, providing higher phylogenetic resolution and revealing finer-scale clustering of the sequences retrieved from the SBR and full-scale EBPR

Environmental Restoration and Remediation↗
PubMed2001

Utilization of evidence-based teaching in U.S. dental hygiene curricula.

PURPOSE: The purpose of this research was to survey U.S. dental hygiene program directors to determine: (1) demographic information, (2) specific Evidence-Based (EB) student instruction methods used, (3) if and how programs use an EB philosophy, (4) perceptions of faculty skills in incorporating EB instruction, and (5) opinions and attitudes regarding future need to incorporate EB philosophies in dental hygiene education. METHODS: Data were gathered by surveying all 235 United States dental hygiene program directors in 1999. The survey included 20 closed items and 1 open-ended item. Initially, the survey was pilot tested using a convenience sample of seven U.S. dental hygiene program directors. A final, revised survey was mailed to the cohort population. A response rate of 70% (n = 164) was achieved after two mailings, and responses were analyzed using descriptive statistics. RESULTS: The demographic results of this study revealed the majority of respondents were from associate degree/certificate dental hygiene programs (77%). Results revealed that most dental hygiene programs are beginning to include some fundamental EB concepts and skills into their curriculum, primarily by incorporating analysis of scientific literature. Most programs provide students with formal library orientation (88%), instruct students in the use of library indices or library databases (86%), and teach the use of the Internet for conducting literature searches (79%). Respondents indicated the major barriers for fully incorporating an EB approach in their dental hygiene program were: lack of faculty skills (37%), no available time (34%), lack of financial resources (33%), and lack of technical support (28%). CONCLUSION: Findings of this study suggest dental hygiene educators have made small strides in creating an EB philosophy dental hygiene curriculum. However, the future of dental hygiene education must address the need for faculty development and training in areas such as computer utilization in core dental hygiene courses, strategies to improve the curriculum to stimulate students' critical thinking skills, and to develop educators' skills in the use of evidence for clinical decision-making.

Attitude of Health Personnel↗
PubMed1992

[Opinion of family and community medicine resident physicians in Spanish hospitals].

OBJECTIVE: To find out the opinions of Family and Community medicine interns concerning various aspects of their teaching during their period of hospital training. DESIGN: Crossover study. SITE. Within hospitals. PARTICIPANTS: Family and Community Medicine residential interns in Spanish hospitals. MAIN MEASUREMENTS AND RESULTS: Out of the questionnaires returned by mail, 78.4% thought that their hospital training was adequate; 83.6% believed they were well enough trained to practise professionally; 76% considered that their hospital's technical equipment had been useful for their training; 66% felt they had sufficient back-up from the staff; 60% contributed to scientific statements or publications; 59% thought that their centre's library was inadequately equipped; and 45.4% stated that their pay as interns, or in the future as specialists, was too low. CONCLUSIONS: Family and Community Medicine interns' current training is in general satisfactory. Certain aspects, such as stocking libraries, salaries and contributions to scientific reports should be improved.

Adult↗
PubMed1998

Bacterial density and survey of cultivable heterotrophs in the surface water of a freshwater marsh habitat of Anopheles quadrimaculatus larvae (Diptera: Culicidae).

We examined surface water samples collected in September and October 1994 from a freshwater marsh habitat containing larval Anopheles quadrimaculatus mosquitoes. Bacterial densities in direct microscopic counts ranged from 9.7 x 10(5) to 1.3 x 10(7) cells/ml. Densities of cultivable bacteria on trypticase soy agar medium ranged from 1.0 to 1.5 x 10(5) cells/ml. The majority of 888 isolates were gram-positive rods (41%) followed by gram-negative rods (28%). Analysis of the cellular fatty acid profiles of 824 isolates using gas chromatography and Microbial Identification Systems TSBA (Rev. 3.60) library software grouped the bacteria into Bacillus spp. (35%), other gram-positive bacteria (16%), pseudomonads (15%), other gram-negative bacteria including mainly Enterobacteriaceae and Vibrionaceae (21%), and profiles not recognized (13%). Among 33 genera within these groups, the most common were Bacillus, Pseudomonas, Aeromonas, and Arthrobacter.

Animals↗
PubMed2001

From Arabidopsis to rice genomics: a survey of French programmes.

During the last ten years, Arabidopsis thaliana has become the most favoured plant system for the study of many aspects of development and adaptation to adverse conditions and diseases. The sequencing of the Arabidopsis thaliana genome is nearly completed with more than 90% of the sequence being released in public databases. This is the first plant genome to be analysed and it has revealed a tremendous amount of information about the nature of the genes it contains and its largely duplicated organisation. French groups have been involved in Arabidopsis genomics at several steps: EST (expressed sequence tags) sequencing, construction and ordering (physical mapping of chromosomes) of a YAC (yeast artificial chromosomes) library, genomic sequencing. In parallel an extensive programme of functional genomics is being undertaken through the systematic analysis of insertional mutants. This information provides a support for analysing other more economically important plant genomes such as the rice genome and constitutes the beginning of a systematic investigation on plant gene functions and will promote new strategies for plant improvement.

Arabidopsis↗
PubMed2006

Tuberculosis of the thyroid gland: review of the literature.

OBJECTIVE: Thyroid tuberculosis (TTB) is a very rare condition, even in countries with a high prevalence of tuberculosis. The aim of this article is to review and retrieve data about thyroid tuberculosis from the English-language literature in order to gain a better understanding of the clinical characteristics of TTB. STUDY DESIGN: We performed Medline, PubMed, and library searches using the key words "thyroid tuberculosis," "throid disease," "tuberculosis and thyroid." Reference lists of the articles obtained and previous reviews were also examined. RESULTS: We retrieved 76 cases matching our selection criteria from the search. Review of the cases with TTB revealed a slight female preponderance. The patients reported in the literature ranged in age from 9 to 83 years, with a median age of 40+/-16 years for men and 44+/-17 years for women. In the articles surveyed, TTB presented with a broad spectrum of manifestations, ranging from an isolated nodule to thyrotoxicosis. It seems that diagnosis of thyroid tuberculosis has recently been increasing, perhaps because of the growing incidence of tuberculosis and the diagnostic use of fine-needle aspiration cytology in the diagnosis. Although, in the past, the diagnosis was generally made by the examination of the specimens, at present, fine-needle aspiration cytology seems to be a useful method in diagnosis tuberculous thyroiditis. The role of surgery is limited after the diagnosis. The choice of treatment should be medical antituberculous therapy. CONCLUSIONS: Preoperative diagnosis of thyroid tuberculosis is important because of the availability of medical treatment and the limited role of surgery. This condition should be kept in mind in evaluating patients with a thyroid nodule, in communities where the prevalence of tuberculosis is high.

Adolescent↗
PubMed2003

Delimitation of the fertility restorer locus Rfk1 to a 43-kb contig in Kosena radish (Raphanus sativus L.).

We are pursuing a positional cloning strategy to isolate the fertility restoration gene Rfk1 from radish. Random polymorphic DNA-sequence-tagged site (RAPD-STS) markers tightly linked to the gene in radish were isolated, and a RAPD map surrounding the Rfk1 locus was constructed. We surveyed 948 F2 plants with adjacent RAPD-STS markers to isolate recombinants for bulk segregant analysis. This analysis was effective in isolating tightly linked amplification fragment length polymorphism (AFLP) markers surrounding the gene of interest. Ten tightly linked AFLP markers were obtained and used to construct a high-resolution map of the region. The closest AFLP-STS markers flanking Rfk1 were 0.1 cM and 0.2 cM away. Using the four adjacent AFLP markers, we screened lambda and cosmid libraries. The lambda and cosmid clones were aligned by examination of end sequences and restriction fragment length polymorphism (RFLP) patterns for each clone, and by hybridization to the DNA isolated from recombinants. Finally, we constructed a 198-kb contig encompassing the Rfk1 gene and comprising 20 lambda and two cosmid clones. By analysis of the breakpoints in recombinants with the rfk1/rfk1 or Rfk1/- genotype, the Rfk1 locus could be assigned to a 43-kb region comprising four lambda clones and one cosmid clone. This pinpoint localization in the radish genome has made it possible for us to identify the gene by sequence analysis and genetic transformation of cytoplasmic male-sterile Brassica napus plants.

Blotting, Southern↗
PubMed2006

Identification and analysis of genes expressed in the adult filarial parasitic nematode Dirofilaria immitis.

The heartworm Dirofilaria immitis is a filarial parasitic nematode infecting dogs and other mammals worldwide causing fatal complications. Here, we present the first large-scale survey of the adult heartworm transcriptome by generation and analysis of 4005 expressed sequence tags, identifying about 1800 genes and expanding the available sequence information for the parasite significantly. Brugia malayi genomic data offered the most valuable information to interpret heartworm genes, with about 70% of D. immitis genes showing significant similarities to the assembly. Comparative genomic analyses revealed both genes common to metazoans or nematodes and genes specific to filarial parasites that may relate to parasitism. Characterization of abundant transcripts suggested important roles for genes involved in energy generation and antioxidant defense in adults. In particular, we proposed that adult heartworm likely adopted an anaerobic electron transfer-based energy generation system distinct from the aerobic pathway utilized by its mammalian host, making it a promising target in developing next generation macrofilaricides and other treatments. Our survey provided novel insights into the D. immitis transcriptome and laid a foundation for further comparative studies on biology, parasitism and evolution within the phylum Nematoda.

Animals↗
PubMedDate not supplied

Paternity exclusion in koalas using hypervariable microsatellites.

Koala microsatellite loci containing the dinucleotide motif (CA)n were isolated from a size-fractionated (250-500 bp) koala genomic library and sequenced. Six locus-specific primer pairs were designed and synthesized for DNA amplification using the polymerase chain reaction (PCR). Microsatellite genotyping of 12 individuals generated unique "fingerprints" for each koala. All six microsatellite loci were polymorphic, with a mean of 6.5 +/- 0.6 alleles/locus. This level of allelic diversity is capable of generating > 4 x 10(9) DNA profiles, making it the most powerful technology for fingerprinting koalas currently available. Observed heterozygosities (H(o)) in the eight unrelated individuals surveyed ranged from 0.25 to 0.75, with a mean of 0.54 +/- 0.06. Mendelian inheritance of the observed polymorphism was confirmed by family studies. We demonstrate that microsatellite loci are ideal genetic markers for paternity exclusion and pedigree analysis of koalas, which have shown little genetic variation using most other methods.

Animals↗
PubMed1991

delta 4-3-Oxosteroid 5 beta-reductase. Structure and function.

delta 4-3-Oxosteroid 5 beta-reductase catalysing reduction of delta 4-3-oxosteroids to give A/B cis-conformation was intraperitoneally injected into BALB/c strain mice with Ribi adjuvant. Monoclonal antibody specific for this enzyme was prepared from their spleen cells. Using this monoclonal antibody as a probe the enzyme was further purified using reversed phase liquid chromatography to determine amino-acid sequence protein-chemically. Attempts to determine the N-terminal amino acid failed, indicating that the N-terminal amino acid is blocked. The protein was therefore subjected to digestion with lysyl endopeptidase after alkylating with iodoacetate. The peptides thus formed were isolated and purified by reversed-phase high-performance liquid chromatography and their amino-acid sequences were determined. Using antibodies and oligonucleotides as probes a cDNA which contained a 978 bp long open reading frame encoding 326 amino-acid residues (Mr 37376) was isolated from rat liver cDNA libraries and the entire sequence of the protein was deciphered from its nucleotide sequence. The COS cells transfected with this cDNA revealed a versatile activity to reduce varied kinds of delta 4-3-oxosteroids, i.e. 7 alpha-hydroxy-4-cholesten-3-one, androstenedione and cortisone as postulated by Okuda and Okuda (1984, J. Biol. Chem. 259, 7519-7524) and Furuebisu et al. (1987, Biochim. Biophys. Acta 912, 110-114. With a newly established immunoblotting assay method several tissues and organs were surveyed and it was found that the enzyme exists only in the liver and there is an apparent difference between sexes as to the content of this enzyme. However, there was little if any difference in the amount of mRNAs between both sexes, which may indicates that the sexual difference of rat liver cytosol 5 beta-reductase is due to a posttranslational modification and/or degradation.

Amino Acid Sequence↗
PubMedDate not supplied

Learner support experienced by RNs in a collaborative distance RN-to-BSN program.

Sixteen RN-to-BSN students participated in interviews at the start and at the completion of taking their first class from a newly implemented, university-based, collaborative distance learning program. The purpose of the taped, semi-structured telephone interviews was to identify the support RNs experienced. Institutional themes of support that emerged were orientation programs, technical support, mentors, library resources, and prompt feedback from instructors. Non-institutional support themes focused on family, friends, peers, the workplace, and learners.

Adult↗
PubMed1992

Increases in knowledge and use of information technology by entering medical students at McMaster University in successive annual surveys.

OBJECTIVE: To determine self-reported microcomputer and information technology competency, access, and usage by entering medical students and their perceptions of the need for training in additional applications. DESIGN: Cross-sectional surveys of successive classes. SETTING: McMaster University Faculty of Health Sciences Medical Undergraduate Program, which has a 33-month, problem-based, self-directed learning curriculum and a high applicant-to-student ratio. PARTICIPANTS: Medical school classes entering in 1987, 1988, and 1989. Response rates were 80%, 90%, and 86% respectively. MEASURES: A self-report questionnaire was sent to each student, with up to two follow-up letters to prompt a response. RESULTS: There was a progressive rise in reported information technology access and use for the three years. For the classes starting in 1987, '88, and '89 respectively, computer access was 29%, 49%, and 49% (P < 0.002 for linear trend), and, among those with computer access, modem access was 17%, 29% and 50% (P = 0.012). Self-service MEDLINE use on CD-ROM at the Health Sciences Library was 65%, 75%, and 89% respectively (P < 0.001) for all respondents within the first few months of starting medical school. Over 50% of each class stated they would take courses, if available, on clinical applications software, office management, online searching, filing, and CD-ROM searching. CONCLUSIONS: Half of the most recent entering students already had access to a personal computer and most wished to learn computer applications that would assist them with patient management, and with information access and organization. Medical schools need to address which applications they will teach or make available and how to bring all students to acceptable competency in their use.

Computer Literacy↗
PubMed1997

Decidual/trophoblast prolactin-related protein: characterization of gene structure and cell-specific expression.

Decidual/trophoblast PRL-related protein (d/tPRP) is a member of the PRL gene family and is dually expressed in uterine and placental tissues in a highly coordinated pattern during pregnancy. In the present study, we describe the isolation and characterization of the d/tPRP gene. A lambda DASH II Wistar-Kyoto rat genomic library was screened with a labeled d/tPRP complementary DNA, resulting in the isolation of two phage clones, RGLd-41 [17.7 kilobases (kb)] and RGLd-42 (15.8 kb). RGLd-41 alone was found to contain the full-length d/tPRP gene and was used for subsequent analyses. The d/tPRP gene possesses a six-exon, five-intron organization. Relative to other highly conserved members of the PRL gene family, d/tPRP contains a single small additional exon (exon 3) situated between exons 2 and 3 of the prototypical PRL gene. The region corresponding to exon 3 of d/tPRP encodes for a unique amino acid region found in a subset of PRL family members. A reverse transcription-PCR (RT-PCR) tissue survey for d/tPRP messenger RNA revealed that d/tPRP expression was restricted to decidual and trophoblast tissues. A single transcription start site 65 bp upstream of the initiation codon was identified in decidual tissue, whereas multiple transcription start sites ranging from 61-66 bp upstream of the initiation codon were detected in placental tissue. Various tissue culture systems (primary cultures and cell lines) were evaluated for d/tPRP expression and activation of a 3.96-kb d/tPRP promoter-luciferase reporter construct. Decidual, spongiotrophoblast, and trophoblast giant cell populations expressed d/tPRP and were capable of activating the d/tPRP promoter-reporter construct, whereas other cell types were ineffective. Limited d/tPRP promoter activation was noted in uterine stromal cell lines. In summary, d/tPRP possesses a unique six-exon, five-intron gene structure and exhibits cell-specific expression that is regulated at least in part by a 3.96-kb 5'-flanking region.

Amino Acid Sequence↗
PubMed2000

PPARgamma: observations in the hematopoietic system.

Human Peroxisome Proliferator-Activated Receptor gamma (PPARgamma) was originally cloned from a human bone marrow library. What role does this ligand activated transcription factor play in hematopoiesis and the immune system? We note that: a) PPARgamma has potential to interact/interfere or synergize with retinoid biology, b) fatty acids and a prostaglandin have been identified as ligands, and c) lymphocytes, monocytes and neutrophils use fatty acids as a major source of energy production, d) PPARgamma has been shown to oppose TNFalpha and down regulate cytokine production in monocytes. Therefore, we undertook a review of the literature and an expression survey of PPARgamma in a number of major organs and cells involved in the hematopoietic system, for the purpose of building a database towards understanding the role and function of PPARgamma gene regulation in the developing blood and immune systems. PPARgamma is expressed before mesodermal induction in tissue in and around Speymann's organizer in the xenopus blastocyst, in erythroid precursors of blood islands and in the circulation of the day 10.0 murine embryo, in human 19 week fetal liver, in some but not all murine and human bone marrow erythroid, myeloid, and monocytoid progenitors, bone marrow stromal cells and adipocytes, osteoblasts, endothelial cells, some T, and B lymphocytes, monocytes, macrophages, and other monocytic derivatives. It can be found in the cells of Peyer's patches, lymphoid follicles, spleen, and thymus. It is not clear if it is ever or transiently expressed in megakaryocytes, mast cells, or neutrophils. Based on the above data and a review of the literature, PPARgamma seems to play a role during the elicitation of immune responses. We propose PPARgamma may be involved in changes in energy states required during activation and development of many cell types involved, and has additional immunologically relevant effects in erythroid, myeloid, monocytic, T and B lymphocytic, stromal, and endothelial cell function.

Amino Acid Sequence↗
PubMed2003

Obstetricians' attitudes, subjective norms, perceived controls, and intentions on antibiotic prophylaxis in caesarean section.

Over 10% of current births in all countries of the world are delivered by caesarean section. Single-dose ampicillin or cefazolin administered after cord clamping has been proven to be effective for the prevention of post-caesarean infections as indicated in many randomised trials and reviews in the Cochrane Library. This study aimed to determine three determinants of behavioural intention using the theory of planned behaviour: attitudes, subjective norms, and perceived controls. Intentions were examined for five aspects of the use of antibiotic prophylaxis, namely whether or not antibiotics were used, used in all caesarean sections, after rather than before cord clamping, whether ampicillin/cefazolin or broader-spectrum antibiotics were used, and whether single or multiple doses were given. Fifty obstetricians selected from university, regional, and general hospitals in southern Thailand, were surveyed using a questionnaire and in-depth interview. Their intentions to use a single dose and to use in all cases were low, and this was related to negative attitudes and reference groups who did not approve of the single dose. The negative attitude was based on scepticism concerning the applicability of well-equipped trials from the developed world and fear of consequences of post-caesarean infections. Norms carried over from residency training had more long-term influence in their practice than newer information from books or journals. Perceived external controls on their practice were less predictive of intentions. Intentions were only partly predictive of behaviour. Changing attitudes, introducing evidence-based information into residency training and strengthening control systems in the hospital are essential to improve intentions.

Adult↗
PubMed1994

Comparative study of cellulases associated with adventitious root initiation, apical buds, and leaf, flower, and pod abscission zones in soybean.

Cellulase activity was measured in soybean (Glycine max) leaf abscission zones, flower abscission zones, pod abscission zones, apical buds, and adventitious rooting hypocotyls. Immunoprecipitation data showed that a cellulase immunologically similar to the bean abscission cellulase (isoelectric point 9.5) is present in soybean leaf, flower, and pod abscission zones, but is not present in soybean apical buds or rooting hypocotyls. cDNA and genomic clones for two different soybean genes were identified and show sequence similarity with the bean abscission cellulase clone pBAC10. The cDNA clone pSAC1, isolated from a soybean abscission cDNA library, hybridized to transcripts in soybean leaf, flower, and pod abscission zones. Although ethylene has been shown to play a role in the increase in cellulase activity associated with both abscission and adventitious root initiation, no signal was seen for hybridization of the soybean abscission cellulase clone, pSAC1, to RNA from soybean adventitious rooting hypocotyls. In addition, no soybean abscission cellulase transcripts were detected in apical buds. Transcripts for a second soybean cellulase gene (SC2) were not detected in any of the tissues surveyed.

Amino Acid Sequence↗
PubMed2000

Lysine 2,3-aminomutase from Clostridium subterminale SB4: mass spectral characterization of cyanogen bromide-treated peptides and cloning, sequencing, and expression of the gene kamA in Escherichia coli.

Lysine 2,3-aminomutase (KAM, EC 5.4.3.2.) catalyzes the interconversion of L-lysine and L-beta-lysine, the first step in lysine degradation in Clostridium subterminale SB4. KAM requires S-adenosylmethionine (SAM), which mediates hydrogen transfer in a mechanism analogous to adenosylcobalamin-dependent reactions. KAM also contains an iron-sulfur cluster and requires pyridoxal 5'-phosphate (PLP) for activity. In the present work, we report the cloning and nucleotide sequencing of the gene kamA for C. subterminale SB4 KAM and conditions for its expression in Escherichia coli. The cyanogen bromide peptides were isolated and characterized by mass spectral analysis and, for selected peptides, amino acid and N-terminal amino acid sequence analysis. PCR was performed with degenerate oligonucleotide primers and C. subterminale SB4 chromosomal DNA to produce a portion of kamA containing 1,029 base pairs of the gene. The complete gene was obtained from a genomic library of C. subterminale SB4 chromosomal DNA by use of DNA probe analysis based on the 1,029-base pair fragment. The full-length gene consisted of 1,251 base pairs specifying a protein of 47,030 Da, in reasonable agreement with 47, 173 Da obtained by electrospray mass spectrometry of the purified enzyme. N- and C-terminal amino acid analysis of KAM and its cyanogen bromide peptides firmly correlated its amino acid sequence with the nucleotide sequence of kamA. A survey of bacterial genome databases identified seven homologs with 31 to 72% sequence identity to KAM, none of which were known enzymes. An E. coli expression system consisting of pET 23a(+) plus kamA yielded unsatisfactory expression and bacterial growth. Codon usage in kamA includes the use of AGA for all 29 arginine residues. AGA is rarely used in E. coli, and arginine clusters at positions 4 and 5, 25 and 27, and 134, 135, and 136 apparently compound the barrier to expression. Coexpression of E. coli argU dramatically enhanced both cell growth and expression of KAM. Purified recombinant KAM is equivalent to that purified from C. subterminale SB4.

Amino Acid Sequence↗