Structure of genes encoding steroidogenic enzymes

Synthesis of adrenal steroid hormones from cholesterol entails the actions of only five enzymes, four of which are specific forms of cytochrome P450. These cytochrome P450 enzymes have all been isolated and their activities reconstituted in vitro, showing that each enzyme catalyses multiple steroidal conversions. Genes or complementary DNAs have been cloned for human P450scc (the cholesterol side-chain cleavage enzyme), P450c17 (17 alpha-hydroxylase/17,20 lyase) and P450c21 (21-hydroxylase). The sequences for microsomal P450c17 and P450c21 are much more closely related to one another than either is to the sequence for mitochondrial P450scc. Each of these P450 enzymes is encoded by a single human gene; the gene for P450scc lies on chromosome 15, that for P450c17 lies on chromosome 10, and that for P450c21 lies on chromosome 6. The human, mouse and bovine genomes each have two P450c21 genes. While only one of these is active in mouse and man, both genes may be active in cattle. A wide variety of lesions in the human P450c21(B) gene causes congenital adrenal hyperplasia, a common genetic disorder.     

Oligonucleotide primers for detection and amplification of the emoA gene encoding bacterial ethylenediaminetetraacetate monooxygenase

A system of primers was designed on the basis of analysis of nucleotide sequences of the emoA gene encoding ethylenediaminetetraacetate (EDTA) monooxygenase, which are deposited in GenBank. This system of primers makes it possible to obtain emoA gene fragments approximately 750 bp long for bacterial destructors of EDTA. Polymerase chain reaction (PCR) of total DNA isolated from enrichment and pure cultures showed that this system can be effectively used for detecting the emoA gene in representatives of Alpha- and Gammaproteobacteria. Partial sequences of emoA genes of bacteria of the genera Chelativorans and Stenotrophomonas, which are able to degrade this pollutant, have been sequenced and deposited in GenBank.     

SNP detection and genetic mapping of porcine genes encoding enzymes in hepatic metabolic pathways and evaluation of linkage with carcass traits

We have previously identified and mapped porcine expressed sequence tags (ESTs) derived from genes that are preferentially expressed in liver. The aim of the present study was to identify single nucleotide polymorphisms (SNPs) in porcine genes encoding enzymes in hepatic metabolic pathways and use the SNPs for mapping. Furthermore, these genes, which are involved in utilization and partitioning of nutrients, were examined for their effects on carcass and meat quality traits by linkage analyses. In total, 100 ESTs were screened for SNPs by single strand conformation polymorphism analyses across a diverse panel of animals with a 36% success rate. Twelve of 36 polymorphic loci segregated in a three-generation Duroc x Berlin Miniature Pig (F2) resource population, the DUMI resource population, and were genetically mapped. Interval mapping of the corresponding chromosomes was performed to verify mapping of the genes within quantitative trait loci (QTL) regions detected in this resource population. QTL with genome-wide significance were detected in the vicinity of GNMT, ESTL147 and HGD. These loci therefore are positional candidate genes.     

Molecular ecology of tetracycline resistance: development and validation of primers for detection of tetracycline resistance genes encoding ribosomal protection proteins

Phylogenetic analysis of tetracycline resistance genes encoding the ribosomal protection proteins (RPPs) revealed the monophyletic origin of these genes. The most deeply branching class, exemplified by tet and otrA, consisted of genes from the antibiotic-producing organisms Streptomyces rimosus and Streptomyces lividans. With a high degree of confidence, the corresponding genes of the other seven classes (Tet M, Tet S, Tet O, Tet W, Tet Q, Tet T, and TetB P) formed phylogenetically distinct separate clusters. Based on this phylogenetic analysis, a set of PCR primers for detection, retrieval, and sequence analysis of the corresponding gene fragments from a variety of bacterial and environmental sources was developed and characterized. A pair of degenerate primers targeted all tetracycline resistance genes encoding RPPs except otrA and tet, and seven other primer pairs were designed to target the specific classes. The primers were used to detect the circulation of these genes in the rumina of cows, in swine feed and feces, and in swine fecal streptococci. Classes Tet O and Tet W were found in the intestinal contents of both animals, while Tet M was confined to pigs and Tet Q was confined to the rumen. The tet(O) and tet(W) genes circulating in the microbiota of the rumen and the gastrointestinal tract of pigs were identical despite the differences in animal hosts and antibiotic use regimens. Swine fecal streptococci uniformly possessed the tet(O) gene, and 22% of them also carried tet(M). This population could be considered one of the main reservoirs of these two resistance genes in the pig gastrointestinal tract. All classes of RPPs except Tet T and TetB P were found in the commercial components of swine feed. This is the first demonstration of the applicability of molecular ecology techniques to estimation of the gene pool and the flux of antibiotic resistance genes in production animals.     

Regulation of expression of the genes encoding steroidogenic enzymes

In recent years it has become apparent that tropic hormones involved in steroidogenesis act to regulate the expression of the enzymes involved in the various steroidogenic pathways. This is particularly evident in the ovary where the episodic secretion of steroids throughout the ovarian cycle is regulated largely by changes in the levels of the particular enzymes involved in each step of the steroid biosynthetic pathways. Recently, the genes for the various cytochrome P450 species involved in ovarian steroidogenesis, namely cholesterol side-chain cleavage P450 (P450SCC), 17 alpha-hydroxylase P450 (P450(17 alpha], and aromatase cytochrome P450 (P450AROM) have been isolated and characterized, making it possible to study the regulation of expression at the molecular level. To this end, a series of chimeric constructs have been prepared in which fragments of the 5'-untranslated region of bovine P450(17 alpha) and P450SCC have been inserted upstream of the chloramphenicol acetyl transferase (CAT) and beta-globin reporter genes. These constructs have been used to transfect primary cultures of bovine luteal and thecal cells. The results indicate that cAMP responsiveness lies within defined regions of genes which do not contain a classical CRE, similar to previous results utilizing adrenal cells in culture. Furthermore, although constructs containing both the P450(17 alpha) and P450SCC 5'-upstream regions are expressed in both luteal and thecal cell cultures, only those containing the P450SCC sequences are expressed in luteal cells. Studies on the expression of P450AROM indicate that the promoter which is responsible for its expression in human placenta is not operative in the corpus luteum. Thus estrogen biosynthesis may be regulated by the differential use of tissue specific promoters, thus accounting for the complexity and multifactorial nature of the expression of this activity.     

A novel transgenic chimaeric mouse system for the rapid functional evaluation of genes encoding secreted proteins

A major challenge of the post-genomic era is the functional characterization of anonymous open reading frames (ORFs) identified by the Human Genome Project. In this context, there is a strong requirement for the development of technologies that enhance our ability to analyze gene functions at the level of the whole organism. Here, we describe a rapid and efficient procedure to generate transgenic chimaeric mice that continuously secrete a foreign protein into the systemic circulation. The transgene units were inserted into the genomic site adjacent to the endogenous immunoglobulin (Ig) κ locus by homologous recombination, using a modified mouse embryonic stem (ES) cell line that exhibits a high frequency of homologous recombination at the Igκ region. The resultant ES clones were injected into embryos derived from a B-cell-deficient host strain, thus producing chimaerism-independent, B-cell-specific transgene expression. This feature of the system eliminates the time-consuming breeding typically implemented in standard transgenic strategies and allows for evaluating the effect of ectopic transgene expression directly in the resulting chimaeric mice. To demonstrate the utility of this system we showed high-level protein expression in the sera and severe phenotypes in human EPO (hEPO) and murine thrombopoietin (mTPO) transgenic chimaeras.     

Designing universal primers for the isolation of DNA sequences encoding Proanthocyanidins biosynthetic enzymes in Crataegus aronia

ABSTRACT: BACKGROUND: Hawthorn is the common name of all plant species in the genus Crataegus, which belongs to the Rosaceae family. Crataegus are considered useful medicinal plants because of their high content of proanthocyanidins (PAs) and other related compounds. To improve PAs production in Crataegus tissues, the sequences of genes encoding PAs biosynthetic enzymes are required. FINDINGS: Different bioinformatics tools, including BLAST, multiple sequence alignment and alignment PCR analysis were used to design primers suitable for the amplification of DNA fragments from 10 candidate genes encoding enzymes involved in PAs biosynthesis in C. aronia. DNA sequencing results proved the utility of the designed primers. The primers were used successfully to amplify DNA fragments of different PAs biosynthesis genes in different Rosaceae plants. CONCLUSION: To the best of our knowledge, this is the first use of the alignment PCR approach to isolate DNA sequences encoding PAs biosynthetic enzymes in Rosaceae plants.     

System of oligonucleotide primers for detection and amplification of the emoA gene encoding bacterial ethylenediaminetetraacetate monooxygenase

A system of primers was designed on the basis of analysis of nucleotide sequences of the emoAgene encoding ethylenediaminetetraacetate (EDTA) monooxygenase, which are deposited in GenBank. This system of primers makes it possible to obtain emoA gene fragments approximately 750 bp long for bacterial destructors of EDTA. Polymerase chain reaction (PCR) of total DNA isolated from enrichment and pure cultures showed that this system can be effectively used for detecting the emoAgene in representatives of Alpha- and Gammaproteobacteria. Partial sequences of emoA genes of bacteria of the genera Chelativorans and Stenotrophomonas, which are able to degrade this pollutant, have been sequenced and deposited in GenBank.     

Design and in vitro evaluation of new rpoB-DGGE primers for ruminants

Two new primer sets based on the rpoB gene were designed and evaluated with bovine and ovine rumen samples. The newly developed rpoB-DGGE primer set was used along with the 16S rRNA gene-V3, and another (old) rpoB-DGGE-based primer set from a previous study to in vitro compare the bovine and ovine rumen ecosystems. The results indicate a significant (P<0.001) difference in the microbial population between the two ruminants irrespective of the primers used in the analysis. Qualitative comparison of the data provides evidence for the presence of similar phyla profiles between the 16S rRNA gene and the newly developed rpoB primers. A comparison between the two rpoB-based primer sets (old and new) showed that the old rpoB-based primers failed to amplify phylum Bacteroidetes (a common phylum in the rumen) in both bovine and ovine rumen samples. The old and new rpoB-DGGE-based primers amplified a large number of clones belonging to phylum Proteobacteria, providing a useful insight into the microbial structure of the rumen. ChaoI, ACE, Simpson, and Shannon-Weaver index analysis estimated the bovine rumen to be more diverse than the ovine rumen for all three primer sets. These results provide a new insight into the community structure among ruminants using the newly developed primers in this study.     

Aminoglycoside-inactivating enzymes in strains of Salmonella and genes encoding them

Aminoglycoside resistance patterns of 56 strains isolated from man, cattle and environment were determined. 34 out of 42 gentamicin-resistant strains were shown to produce AAC(3)-II and 7 strains produced ANT(2"). All the 48 kanamycin resistant strains produced APH(3')-I. Spot hybridization of the 42 gentamicin resistant strains with the inner fragment of the aacC2 gene revealed positive signals for all the strains. Hybridization of the 48 kanamycin-resistant strains with the aphA1 gene probe provided positive results in all the strains. The AAC(3)-IV encoding gene was not detected by DNA-DNA hybridization in the strains studied.     

Design and evaluation of malolactic enzyme gene targeted primers for rapid identification and detection of Oenococcus oeni in wine

Rapid identification and detection of Oenococcus oeni was achieved by species-specific PCR. Two primers flanking a 1025 bp region of the O. oeni gene encoding the malolactic enzyme were designed. The expected DNA amplificate was obtained only when purified DNA from O. oeni was used. The identity of PCR product was confirmed by nested PCR and restriction analysis. Within 8 h, 10³ cfu ml⁻¹ of oenococci were detected in fermenting grape must containing 10⁷ yeast cells, whereas the detection limit in wine was 10⁴ cfu ml⁻¹. The rapidity and reliability of the PCR procedure established suggests that the method may be profitably applied in winery laboratories for quality control.     

Chromosomal assignments for porcine genes encoding enzymes in hepatic metabolic pathways

Increasing the number of mapped genes will facilitate (1) the identification of potential candidate genes for a trait of interest within quantitative trait loci regions and (2) comparative mapping. The metabolic activities of the liver are essential for providing fuel to peripheral organs, for regulation of amino acid, carbohydrate and lipid metabolism and for homoeostasis of vitamins, minerals and electrolytes. We aimed to identify and map genes coding for enzymes active in the liver by somatic cell genetics in order to contribute to the improvement of the porcine gene map. We mapped 28 genes of hepatic metabolic pathways including six genes whose locations could be confirmed and 22 new assignments. Localization information in human was available for all but one gene. In total 24 genes were assigned to in the expected chromosomal regions on the basis of the currently available information on the comparative human and pig map while for four genes our results suggest a new correspondence or extended regions of conservation between porcine and human chromosomes.     

Rapid detection and identification of the metabolically diverse genus Gordonia by 16S rRNA-gene-targeted genus-specific primers

The importance of the emerging genus Gordonia in industrial and environmental biotechnology is evidenced by the recent increase in associated publications and patents. But, investigations into potentially valuable Gordonia members are restricted by the limitations of current isolation and detection techniques. This motivated us to design a genus-specific oligonucleotide primer pair which could assist in rapid detection of species of the genus Gordonia by means of PCR-specific amplification. The Gordonia-specific 16S rDNA fragment (829 bp) was successfully amplified for all the reference Gordonia species with the designed primer pair G268F/G1096R. No amplification was noted for closely related species from other genera. The genus specificity was validated with 47 strains including wild-type isolates. Interestingly, two strains assigned earlier as Gordonia nitida (DSM 777) and Gordonia rubripertinctus (ATCC 21930) failed to produce a Gordonia-specific fragment with this primer pair. Further analysis of these two isolates based on 16S rDNA sequencing and phylogenetic analysis classified them to the genus Rhodococcus. Preliminary screening of soil samples with the Gordonia-specific primers was successful in terms of the rapid detection of nine Gordonia wild-type isolates.     

A new method for the rapid identification of genes encoding restriction and modification enzymes.

We have constructed derivatives of Escherichia coli that can be used for the rapid identification of recombinant plasmids encoding DNA restriction enzymes and methyltransferases. The induction of the DNA-damage inducible SOS response by the Mcr and Mrr systems, in the presence of methylated DNA, is used to select plasmids encoding DNA methyltransferases. The strains of E. coli that we have constructed are temperature-sensitive for the Mcr and Mrr systems and have been further modified to include a lacZ gene fused to the damage-inducible dinD locus of E. coli. The detection of recombinant plasmids encoding DNA methyltransferases and restriction enzymes is a simple, one step procedure that is based on the induction at the restrictive temperature of the lacZ gene. Transformants encoding DNA methyltransferase genes are detected on LB agar plates supplemented with X-gal as blue colonies. Using this method, we have cloned a variety of DNA methyltransferase genes from diverse species such as Neisseria, Haemophilus, Treponema, Pseudomonas, Xanthomonas and Saccharopolyspora.     

Characterization of a novel methanol dehydrogenase in representatives of Burkholderiales: implications for environmental detection of methylotrophy and evidence for convergent evolution

Some members of Burkholderiales are able to grow on methanol but lack the genes (mxaFI) responsible for the well-characterized two-subunit pyrroloquinoline quinone-dependent quinoprotein methanol dehydrogenase that is widespread in methylotrophic Proteobacteria. Here, we characterized novel, mono-subunit enzymes responsible for methanol oxidation in four strains, Methyloversatilis universalis FAM5, Methylibium petroleiphilum PM1, and unclassified Burkholderiales strains RZ18-153 and FAM1. The enzyme from M. universalis FAM5 was partially purified and subjected to matrix-assisted laser desorption ionization-time of fight peptide mass fingerprinting. The resulting peptide spectrum was used to identify a gene candidate in the genome of M. petroleiphilum PM1 (mdh2) predicted to encode a type I alcohol dehydrogenase related to the characterized methanol dehydrogenase large subunits but at less than 35% amino acid identity. Homologs of mdh2 were amplified from M. universalis FAM5 and strains RZ18-153 and FAM1, and mutants lacking mdh2 were generated in three of the organisms. These mutants lost their ability to grow on methanol and ethanol, demonstrating that mdh2 is responsible for oxidation of both substrates. Our findings have implications for environmental detection of methylotrophy and indicate that this ability is widespread beyond populations possessing mxaF, the gene traditionally used as a genetic marker for environmental detection of methanol-oxidizing capability. Our findings also have implications for understanding the evolution of methanol oxidation, suggesting a convergence toward the enzymatic function for methanol oxidation in MxaF and Mdh2-type proteins.     

Heterogeneity of the glutathione transferase genes encoding enzymes responsible for insecticide degradation in the housefly

One of the four glutathione-S-transferases (GST) that is overproduced in the insecticide-resistant Cornell-R strain of the housefly (Musca domestica) produces an activity that degrades the insecticide dimethyl parathion and conjugates glutathione to lindane. In earlier work, it was shown that the resistant Cornell-R carries an amplification, probably a duplication, of one or more of its GST loci and that this amplification is directly related to resistance. Using polymerase chain reaction (PCR) amplification with genomic DNA, multiple copies of the gene encoding the parathion-degrading activity (called MdGst-3) were subcloned from both the ancestral, insecticide-susceptible strain BPM and from the insecticide-resistant Cornell-R. In BPM, three different MdGst-3 genes were identified while in Cornell-R, 12 different MdGst-3 sequences were found that, though closely related to ancestral genes, had diverged by a few nucleotides. This diversity in MdGst-3 genomic sequences in Cornell-R is reflected in the expressed sequences, as sampled through a cDNA bank. Population heterozygosity cannot account for these multiple GST genes. We suggest that selection for resistance to insecticides has resulted in not only amplification of the MdGst-3 genes but also in the divergence of sequence between the amplified copies. Received: 22 November 1995 / Accepted: 23 February 1996