Gene cluster on pAO1 of Arthrobacter nicotinovorans involved in degradation of the plant alkaloid nicotine: cloning, purification, and characterization of 2,6-dihydroxypyridine 3-hydroxylase.

A 27,690-bp gene cluster involved in the degradation of the plant alkaloid nicotine was characterized from the plasmid pAO1 of Arthrobacter nicotinovorans. The genes of the heterotrimeric, molybdopterin cofactor (MoCo)-, flavin adenine dinucleotide (FAD)-, and [Fe-S] cluster-dependent 6-hydroxypseudooxynicotine (ketone) dehydrogenase (KDH) were identified within this cluster. The gene of the large MoCo subunit of KDH was located 4,266 bp from the FAD and [Fe-S] cluster subunit genes. Deduced functions of proteins encoded by open reading frames (ORFs) of the cluster were correlated to individual steps in nicotine degradation. The gene for 2,6-dihydroxypyridine 3-hydroxylase was cloned and expressed in Escherichia coli. The purified homodimeric enzyme of 90 kDa contained 2 mol of tightly bound FAD per mol of dimer. Enzyme activity was strictly NADH-dependent and specific for 2,6-dihydroxypyridine. 2,3-Dihydroxypyridine and 2,6-dimethoxypyridine acted as irreversible inhibitors. Additional ORFs were shown to encode hypothetical proteins presumably required for holoenzyme assembly, interaction with the cell membrane, and transcriptional regulation, including a MobA homologue predicted to be specific for the synthesis of the molybdopterin cytidine dinucleotide cofactor.

Simultaneous identification of mutations by dual-parameter multiplex hybridization in peptide nucleic acid-containing virtual arrays.

The physical entrapment of peptide nucleic acids (PNA) in electrophoresis media provides a system for performing real-time hybridization. DNA strands fully complementary to the target PNA are retarded compared to single-nucleotide mismatched strands. A second parameter, that of amplicon length, has been introduced to perform multiplex analyses on several mutations simultaneously. Size fractionation creates a virtual array of PCR products that can hybridize to one of a set of mutation-specific PNAs present within the matrix. Each targeted mutation can be identified by the size of its corresponding amplicon. Its genotype is characterized by its interaction with a specific PNA that gives a visually resolved distinction between wildtype and mutant allele. In contrast to conventional hybridization, heterozygotes are readily distinguished from homozygotes. Using a capillary electrophoresis-based DNA sequencer, this approach has been used to automate the identification of the H63D, S65C, and C282Y mutations in the hereditary hemochromatosis gene.

Automated detection of point mutations by electrophoresis in peptide-nucleic acid-containing gels.

Polymerization of electronically, essentially neutral peptide nucleic acids (PNA) into polyacrylamide gels creates a medium in which the salt-independent properties of PNA/DNA interactions are used to achieve hybridization with target DNA during affinity electrophoresis. Such physical entrapment of PNA has been used to differentiate between a retarded, complementary DNA strand and a non-retarded sequence differing by a single point mutation. Analysis of fluorescent PCR products--from both model mismatches and clinically relevant point mutations using a conventional automated DNA sequencer--allows one to follow this hybridization in real time and to distinguish homo- and heterozygous mutants visually. It has been shown that parameters affecting the resolution of these species include not only temperature and concentration (as a function of the [GC] content of the PNA), but also the position of the PNA binding sequence within the interacting DNA segment. Under conditions optimized in terms of temperature and PNA concentration, the maximum separation of retarded from non-retarded DNA single strands is obtained when the PNA binding sequence is close to either DNA terminus. Strategies of PNA and PCR primer design that permit a diagnostic application ranging over 85-451 DNA base pairs are proposed.

Variability in the stability of DNA-peptide nucleic acid (PNA) single-base mismatched duplexes: real-time hybridization during affinity electrophoresis in PNA-containing gels.

The stability of all single-base mismatched pairs between a peptide nucleic acid 11-mer and its complementary DNA has been quantified in terms of their melting temperature and compared with the limited amount of data published to date. The strength of the interaction was determined by an automated affinity-electrophoretic approach permitting the visualization, in real time, of hybridization between a physically immobilized peptide nucleic acid and a complementary DNA migrating in an electric field. The dissociation constants are in the range of 10(-7) M (for mismatches) to 10(-10) M (for fully complementary DNA), which are in excellent agreement with solution studies. These and other thermodynamic constants can be accurately, rapidly, and reproducibly measured in this system at concentrations approaching dissociation conditions by using fluorescently labeled DNA in conjunction with commercial DNA sequencers. The stability of single-base mismatched peptide nucleic acid-DNA duplexes depends both on the position as well as on the chemical nature of the mismatch. The stability is at a minimum when the mutation is positioned 4 bases from either terminus (a loss of 20 degreesC or more in the melting temperature) but regains substantial stability when the mismatch is at the center of the duplex. The most stable mismatched pairs are G:T and T:T whereas destabilization is maximal for A:A and G:G. These observations are of significance in the design of probes for detecting mutations by hybridization.

Purification and cDNA cloning of maize HMGd reveal a novel plant chromosomal HMG-box protein with sequence similarity to HMGa.

We have purified the chromosomal high mobility group (HMG) protein HMGd from maize suspension culture cells, determined the N-terminal amino acid (aa) sequence, and isolated the corresponding cDNA. Sequence analysis showed that the cDNA encoded a protein of 126 aa residues with a theoretical mass of 14,104 Da. The protein contains an HMG-box DNA-binding domain and a short acidic C-terminal tail. HMGd is in approx. 65% of its residues identical to maize HMGa, whereas it is only approx. 46% identical to maize HMGcl/2. The differences to the previously reported HMG proteins in aa sequence, in overall charge and in protein size indicate that we have identified a third type of plant chromosomal HMG-box protein belonging to the HMG1 protein family. Immunoblot analysis with a HMGd antiserum reveals that HMGd is expressed in all tissues tested.

IS1473, a putative insertion sequence identified in the plasmid pAO1 from Arthrobacter nicotinovorans: isolation, characterization, and distribution among Arthrobacter species.

A putative insertion sequence (IS1473) has been cloned and sequenced. The 1087-bp element was found between the moaA and the ndhA genes in the upstream region of the nicotine dehydrogenase (ndh) operon in the 160-kb pAO1 plasmid of Arthrobacter nicotinovorans. It is flanked by an imperfect repeat of 33 bp and carries two overlapping open reading frames which, by programmed -1 translational frameshifting, may produce a transposase of 36.735 Da with a pI = 10. 18. The deduced protein is similar to the transposases IS481 and IS1002 from Bordetella and IS476 from Xanthomonas campestris, all members of the IS3 family. The putative insertion element was found as a single copy in the pAO1 plasmid and absent on the chromosome of the A. nicotinovorans genome. Similar sequences were detected by hybridization on total DNA from Arthrobacter globiformis, Arthrobacter ramosus, and Arthrobacter ureafaciens.

Variability in Arabidopsis thaliana chromosomal high-mobility-group-1-like proteins.

The vertebrate high-mobility-group (HMG) protein HMG1 is an abundant non-histone protein which is considered as an architectural element in chromatin. In the monocotyledonous plant maize, four different HMG1-like proteins (HMGa, HMGc1/2, HMGd) have been identified, whereas other eukaryotes usually express only two different proteins of this type. We have examined here the HMG1-like proteins of the dicotyledonous plant Arabidopsis thaliana. The isolation and analysis of cDNAs encoding five different so far uncharacterised HMG1-like proteins (now termed HMG alpha, HMG beta1/2, HMG gamma, HMG delta) from Arabidopsis indicates that the expression of multiple HMG1-like proteins is a general feature of (higher) plants. The Arabidopsis HMG1-like proteins contain an HMG domain as a common feature, but outside this conserved DNA-binding motif the amino acid sequences are significantly different indicating that this protein family displays a greater structural variability in plants than in other eukaryotes. The five HMG1-like proteins were expressed in Escherichia coli and purified. They bind with somewhat different affinity to linear double-stranded DNA. The recognition of DNA structure is evident from their preferential interaction with DNA minicircles relative to linear DNA. Reverse-transcribed PCR suggested that the five HMG1-like genes are simultaneously expressed in Arabidopsis leaves and suspension culture cells.

Substrate properties of fluorescent ribonucleotides in the terminal transferase-catalyzed labeling of DNA sequencing primers.

Terminal deoxynucleotidyltransferase (terminal transferase, E.C. 2.7.7.31) has been used to add a single fluorescent ribonucleotide to the 3' terminus of DNA sequencing primers, thereby creating primers suitable for automated DNA sequence analysis. The previously introduced procedure using fluorescein-UTP for the postsynthetic labeling of primers can, under appropriate reaction conditions, now be extended to commercially available fluorescein-ATP and fluorescein-CTP permitting greater flexibility in primer design. The products of these addition reactions have been shown to provide sequence data qualitatively and quantitatively identical to those obtained with conventional 5'-terminally labeled primers using cycle sequencing conditions in conjunction with an automated sequencer. Ribonucleotide derivatives of four other dyes (coumarin, tetramethylrhodamine, lissamine and Texas Red) were also examined for their potential in the terminal transferase-catalyzed reaction. Whereas coumarin-UTP was efficiently incorporated giving a monoaddition product, the conjugates of all other dyes with ATP, CTP and UTP were extremely poor substrates under all conditions tested.

Chemically synthesised human immunodeficiency virus P7 nucleocapsid protein can self-assemble into particles and binds to a specific site on the tRNA(Lys,3) primer.

The zinc-bound form of the human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein, p7, aggregates into particles visible by electron microscopy. The HIV primer tRNA(Lys,3) forms similar high molecular weight complexes with p7 that are also detected by gel mobility shift assays. RNA oligonucleotides of the three stem-loop structures in tRNA(Lys,3) were assayed for the competitive inhibition of p7-tRNA(Lys,3) binding by the intensities of free tRNA(Lys,3) bands on native gels. This reveals that the p7 binds specifically to the central domain of tRNA(Lys,3) where the D and T psi C loops come together, but not the anticodon stem-loop.

Nonradioactive labeling of RNA.

A modified ribodinucleotide (named pSEEp) has been synthesized using commercially available components on a DNA synthesizer. The presence of a 3'-terminal primary amino group permits its coupling to a range of nonradioactive labels, exemplified here by fluorescein. The product of chemical derivatization of the parent dinucleotide is a good substrate for T4 RNA ligase-mediated coupling to RNA under very mild conditions; the target RNA is not itself subjected to chemical treatment. The well-defined product of this terminal labeling may be analyzed by, for example, the use of a fluorescence-based DNA sequencer. The applications documented serve to demonstrate the power of this approach suggesting that any procedure depending on the detection or targeted tagging of RNA may be adapted to using pSEEp and its derivatives.

Complete sequence of the maize chloroplast genome: gene content, hotspots of divergence and fine tuning of genetic information by transcript editing.

The nucleotide sequence of the chloroplast (cp) DNA from maize (Zea mays) has been completed. The circular double-stranded DNA, which consists of 140,387 base-pairs, contains a pair of inverted repeat regions (IRA and IRB) with 22,748 base-pairs each, which are separated by a small and a large single copy region (SSC and LSC) of 12,536 and 82,355 base-pairs, respectively. The gene content and the relative positions of a total of 104 genes (70 peptide-encoding genes, 30 tRNA genes and four rRNA genes) are identical with the chloroplast DNA of the closely related species rice (Oryza sativa). A detailed analysis of the two graminean plastomes allows the identification of hotspots of divergence which predominate in one region containing a cluster of tRNA genes and in two regions containing degenerated reading frames. One of these length differences is thought to reflect a gene transfer event from the plastome to the nucleus, which is followed by progressive degradation of the respective chloroplast gene resulting in gene fragments. The other divergent plastome region seems to be due to the complete loss of a plastid gene and its functional substitution by a nuclear encoded eukaryotic homologue. The rate of neutral nucleotide substitutions is significantly reduced for protein coding genes located in the inverted repeat regions. This indicates that the existence of inverted repeat regions confers increased genetic stability of the genes positioned in these regions as compared to genes located in the two single copy regions. Editing events cause the primary structures of several transcripts to deviate from the corresponding genomic sequences by C to U transitions. The unambiguous deduction of amino acid sequences from the nucleotide sequences of the corresponding genes is, therefore, not possible. A survey of the 25 editing positions identified in 13 different transcripts of the maize plastome shows that representatives of all protein coding gene classes are subject to editing. A strong bias exists for the second codon position and for certain codon transitions. Based on the number and the codon transition types, and taking into account the frequency of putative editing sites in all peptide encoding genes and unidentified reading frames, a total number of only few more than the experimentally verified 25 editing sites encoded in the maize plastome is estimated. This corresponds to 0.13% of amino acid positions which cannot be derived from the corresponding codons present in the corresponding genes.

Twintrons are not unique to the Euglena chloroplast genome: structure and evolution of a plastome cpn60 gene from a cryptomonad.

Introns within introns (twintrons) are known only from the Euglena chloroplast genome. Twintrons are group II or III introns, into which another group II or III intron has been transposed. In this paper we describe a non-Euglena twintron structure within a plastid-encoded chaperone gene (cpn60) of the cryptomonad alga Pyrenomonas salina. In addition, the evolutionary relationships between members of the Cpn60 protein family are determined. Our findings permit the inclusion of cryptomonad plastomes in phylogenetic studies of intron evolution and present further evidence for the origin of modern plastids from a cyanobacterial ancestor.

Structural analysis and molybdenum-dependent expression of the pAO1-encoded nicotine dehydrogenase genes of Arthrobacter nicotinovorans.

The genes of nicotine dehydrogenase (NDH) were identified, cloned and sequenced from the catabolic plasmid pAO1 of Arthrobacter nicotinovorans. In immediate proximity to this gene cluster is the beginning of the 6-hydroxy-L-niotine oxidase (6-HLNO) gene. NDH is composed of three subunits (A, B and C) of M(r) 30,011, 14,924 and 87,677. It belongs to a family of bacterial hydroxylases with a similar subunit structure; they have molybdopterin dinucleotide, FAD and Fe-S clusters as cofactors. Here the first complete primary structure of a bacterial hydroxylase is provided. Sequence alignments of each of the NDH subunits show similarities to the sequences of eukaryotic xanthine dehydrogenase (XDH) but not to other known molybdenum-containing bacterial enzymes. Based on alignment with XDH it is inferred that the smallest subunit (NDHB) carries an iron-sulphur cluster, that the middle-sized subunit (NDHA) binds FAD, and that the largest NDH subunit (NDHC) corresponds to the molybdopterin-binding domain of XDH. Expression of both the ndh and the 6-hino genes required the presence of nicotine and molybdenum in the culture medium. Tungsten inhibited enzyme activity but not the synthesis of the enzyme protein. The enzyme was found in A. nicotinovorans cells in a soluble form and in a membrane-associated form. In the presence of tungsten the fraction of membrane-associated NDH increased.

The role of RNA editing in conservation of start codons in chloroplast genomes.

Open reading frames (ORFs), encoded by the plastid genomes of tobacco, liverwort, rice and maize were aligned with a view to studying the conservation of translational start and stop codons created by RNA editing of homologous genes. It became evident that most of the homologous ORFs have conserved translation start and stop signals at the gene level. However, some of the ORFs show differences with respect to extensions of their 3' and 5' terminal regions. For example, the proposed N-termini of the ndhD-encoded peptides from different plant species are very variable in length and amino-acid composition. Sequence analysis of ndhD and the corresponding cDNA shows that editing of an ACG triplet in tobacco, spinach and snapdragon leads to the creation of an AUG codon, corresponding to the start codon in other species. Conservation of translational start codons of plastome-encoded genes can, therefore, be achieved by editing of transcripts, and the definition of plastome-encoded ORFs must take potential editing events into consideration.

The smallest known eukaryotic genomes encode a protein gene: towards an understanding of nucleomorph functions.

Cryptomonads are unicellular algae with plastids surrounded by four membranes. Between the two pairs of membranes lies a periplastidal compartment that harbours a DNA-containing organelle, termed the nucleomorph. The nucleomorph is the vestigial nucleus of a phototrophic, eukaryotic endosymbiont. Subcloning of parts of one nucleomorph chromosome revealed a gene coding for an Hsp70 protein. We demonstrate the expression of this nucleomorph protein-coding gene and present a model for protein transport from the host to the endosymbiont compartment.

The irregular chiasm C-roughest locus of Drosophila, which affects axonal projections and programmed cell death, encodes a novel immunoglobulin-like protein.

The axonal projection mutations irregular chiasm C of Drosophila melanogaster comap and genetically interact with the roughest locus, which is required for programmed cell death in the developing retina. We cloned the genomic region in 3C5 by transposon tagging and identified a single transcription unit that produces a major, spatially and temporally regulated mRNA species of approximately 5.0 kb. Postembryonic expression is strong in the developing optic lobe and in the eye imaginal disc. The gene encodes a transmembrane protein of 764 amino acids with five extracellular immunoglobulin-like domains and similarity to the chicken axonal surface glycoprotein DM-GRASP/SC1/BEN. Both known irreC alleles reduce the level of transcription, whereas the roughestCT mutation disrupts the intracellular domain of the protein.

Enzymatic addition of fluorescein- or biotin-riboUTP to oligonucleotides results in primers suitable for DNA sequencing and PCR.

An enzymatic, post-synthetic, non-isotopic modification of oligonucleotides giving primers that are substrates for chain elongation by DNA polymerases is described. It is shown that terminal deoxynucleotidyl transferase incorporates preferentially and almost exclusively a single fluorescein- or biotin-riboUTP at the 3' terminus of oligonucleotides. This one-step procedure, using readily available materials, permits an economical enzymatic labeling of oligonucleotides designed for fluorescence-based or solid-state DNA sequencing or PCR and offers a convenient alternative to chemical modification.