Nucleotide sequence of nine protein-coding genes and 22 tRNAs in the mitochondrial DNA of the sea starPisaster ochraceus

Summary We have cloned and sequenced over 9 kb of the mitochondrial genome from the sea starPisaster ochraceus. Within a continuous 8.0-kb fragment are located the genes for NADH dehydrogenase subunits 1, 2, 3, and 4L (ND1, ND2, ND3, and ND4L), cytochrome oxidase subunits I, II, and III (COI, COII, and COIII), and adenosine triphosphatase subunits 6 and 8 (ATPase 6 and ATPase 8). This large fragment also contains a cluster of 13 tRNA genes between ND1 and COI as well as the genes for isoleucine tRNA between ND1 and ND2, arginine tRNA between COI and ND4L, lysine tRNA between COII and ATPase 8, and the serine (UCN) tRNA between COIII and ND3. The genes for the other five tRNAs lie outside this fragment. The gene for phenylalanine tRNA is located between cytochrome b and the 12S ribosomal genes. The genes for tRNA^glu and tRNA^thr are 3 to the 12S ribosomal gene. The tRNAs for histidine and serine (AGN) are adjacent to each other and lie between ND4 and ND5. These data confirm the novel gene order in mitochondrial DNA (mtDNA) of sea stars and delineate additional distinctions between the sea star and other mtDNA molecules.     

Unexpected diversity of RNase P, an ancient tRNA processing enzyme: Challenges and prospects

For an enzyme functioning predominantly in a seemingly housekeeping role of 5′ tRNA maturation, RNase P displays a remarkable diversity in subunit make-up across the three domains of life. Despite the protein complexity of this ribonucleoprotein enzyme increasing dramatically from bacteria to eukarya, the catalytic function rests with the RNA subunit during evolution. However, the recent demonstration of a protein-only human mitochondrial RNase P has added further intrigue to the compositional variability of this enzyme. In this review, we discuss some possible reasons underlying the structural diversity of the active sites, and use them as thematic bases for elaborating new directions to understand how functional variations might have contributed to the complex evolution of RNase P.     

Examination of protein sequence homologies: V. New perspectives on evolution between bacterial and chloroplast-type ferredoxins inferred from sequence evidence

Summary Sequence homologies among 34 chloroplast-type ferredoxins were examined using a computer program that quantitatively evaluates the extent of sequence similarity as a correlation coefficient. The resultant alignment contains six gaps representing insertions or deletions of some residues, all of which are located such that they precisely preserve the domains of structural fragments as determined by crystallographic data onSpirulina platensis ferredoxin.In the search for any total correlation between the chloroplast-type and 27 bacterial ferredoxins, 1891 comparison matrices prepared for possible combinations indicated that the bacterial basal sequence of 55 residues has been conserved evolutionarily in the chloroplast-type sequences corresponding to residue positions 36–90 ofSpirulina platensis ferredoxin. In addition, the bacterial connector sequence region was found to be conserved. These findings strongly suggest that the bacterial and chloroplast-type ferredoxins descended from a common ancestor, and branched off after the bacterial gene duplication, whereas the chloroplast-type ferredoxins originally were generated by duplicating the already duplicated bacterial gene, i.e., by double-duplication.     

Improved initial yields in C-terminal sequence analysis by thiohydantoin chemistry using purified diphenylphosphoryl isothiocyanate: NMR evidence for a reaction intermediate in the coupling reaction

A thiohydantoin method for C-terminal sequence analysis of proteins on Zitex membranes involves derivatization of the free alpha-carboxyl group with diphenylphosphoryl isothiocyanate (DPPITC) plus treatment with pyridine to form a peptidylthiohydantoin derivative, cleavage of the thiohydantoin (TH) amino acid from the protein with potassium trimethylsilanolate, and identification of the released TH-amino acid by online reversed-phase HPLC. This automated chemistry, which was adapted to the Hewlett-Packard G 1009A sequencer, has been shown to identify two or three cycles on a wide variety of proteins, but suffers from low initial yields and instability of the DPPITC reagent. We report here an improved method for synthesis and purification of DPPITC. With this procedure the DPPITC reagent is a clear liquid that is stable at room temperature under vacuum for more than 9 months or for more than 24 months as a 1.0M solution in benzene at -20 degrees C. Using the purified reagent we were able to more than double the initial yield (from 30.7 to 72.4%) of TH-amino acid from a test protein and substantially decrease sequencer background. Examination of the reaction between DPPITC and the carboxylate of model N-terminally protected dipeptides with 31P NMR provides spectroscopic evidence for a postulated intermediate formed between the DPPITC and the peptide carboxylate. The reaction intermediate provides new insight into the coupling mechanism.     

Structure, organization, and sequence of alpha satellite DNA from human chromosome 17: evidence for evolution by unequal crossing-over and an ancestral pentamer repeat shared with the human X chromosome.

The centromeric regions of all human chromosomes are characterized by distinct subsets of a diverse tandemly repeated DNA family, alpha satellite. On human chromosome 17, the predominant form of alpha satellite is a 2.7-kilobase-pair higher-order repeat unit consisting of 16 alphoid monomers. We present the complete nucleotide sequence of the 16-monomer repeat, which is present in 500 to 1,000 copies per chromosome 17, as well as that of a less abundant 15-monomer repeat, also from chromosome 17. These repeat units were approximately 98% identical in sequence, differing by the exclusion of precisely 1 monomer from the 15-monomer repeat. Homologous unequal crossing-over is suggested as a probable mechanism by which the different repeat lengths on chromosome 17 were generated, and the putative site of such a recombination event is identified. The monomer organization of the chromosome 17 higher-order repeat unit is based, in part, on tandemly repeated pentamers. A similar pentameric suborganization has been previously demonstrated for alpha satellite of the human X chromosome. Despite the organizational similarities, substantial sequence divergence distinguishes these subsets. Hybridization experiments indicate that the chromosome 17 and X subsets are more similar to each other than to the subsets found on several other human chromosomes. We suggest that the chromosome 17 and X alpha satellite subsets may be related components of a larger alphoid subfamily which have evolved from a common ancestral repeat into the contemporary chromosome-specific subsets.     

Avocado cellulase: nucleotide sequence of a putative full-length cDNA clone and evidence for a small gene family

A cDNA library was prepared from ripe avocado fruit (Persea americana Mill. cv. Hass) and screened for clones hybridizing to a 600 bp cDNA clone (pAV5) coding for avocado fruit cellulase. This screening led to the isolation of a clone (pAV363) containing a 2021 nucleotide transcribed sequence and an approximately 150 nucleotide poly(A) tail. Hybridization of pAV363 to a northern blot shows that the length of the homologous message is approximately 2.2 kb. The nucleotide sequence of this putative full-length mRNA clone contains an open reading frame of 1482 nucleotides which codes for a polypeptide of 54.1 kD. The deduced amino acid composition compares favorably with the amino acid composition of native avocado cellulase determined by amino acid analysis. Southern blot analysis of Hind III and Eco RI endonuclease digested genomic DNA indicates a small family of cellulase genes.     

Genomic analysis reveals a duplication of eight rather than seven short consensus repeats in primate CR1 and CR1L: evidence for an additional set shared between CR1 and CR2

We report the discovery of previously unrecognised short consensus repeats (SCRs) within human and chimpanzee CR1 and CR1L. Analysis of available genomic, protein and expression databases suggests that these are actually genomic remnants of SCRs previously reported in other complement control proteins (CCPs). Comparison with the nucleotide motifs of the 11 defined subfamilies of SCRs justifies the designation g-like because of the close similarity to the g subfamily found in CR2 and MCP. To date, we have identified five such SCRs in human and chimpanzee CR1, one in human and chimpanzee CR1L, but none in either rat or mouse Crry in keeping with the number of internal duplications of the long homologous repeat (LHR) found in CR1 and CR1L. In fact, at the genomic level, the ancestral LHR must have contained eight SCRs rather than seven as previously thought. Since g-like SCRs are found immediately downstream of d SCRs, we suggest that there must have been a functional dg set which has been retained by CR2 and MCP but which is degenerate in CR1 or CR1L. Interestingly, dg is also present in the CR2 component of mouse CR1. The degeneration of the g SCR must have occurred prior to the formation of primate CR1L and prior to the duplication events which resulted in primate CR1. In this context, the apparent conservation of g-like SCRs may be surprising and may suggest the existence of mechanisms unrelated to protein coding. These results provide examples of the many processes which have contributed to the evolution of the extensive repertoire of CCPs.     

No evidence for local adaptation in an invasive alien plant: field and greenhouse experiments tracing a colonization sequence

Background and Aims

Local adaptation enables plant species to persist under different environmental conditions. Evolutionary change can occur rapidly in invasive annual species and has been shown to lead to local adaptation. However, the patterns and mechanisms of local adaptation in invasive species along colonization sequences are not yet understood. Thus, in this study the alien annual Impatiens glandulifera was used to investigate local adaptation to distinct habitats that have been consecutively invaded in central Europe.

Methods

A reciprocal transplant experiment was performed using 15 populations from alluvial deciduous forests, fallow meadows and coniferous upland forests, and a greenhouse experiment was performed in which plants from these habitats were grown under treatments reflecting the main habitat differentiators (shade, soil acidity, competition).

Key Results

Biomass production, specific leaf area, plant height and relative growth rate differed between habitats in the field experiment and between treatments in the greenhouse, but not between seed origins. Overall, there was no indication of local adaptation in either experiment.

Conclusions

Since I. glandulifera is a successful invader in many habitats without showing local adaptation, it is suggested that the species is coping with environmental variation by means of high phenotypic plasticity. The species seems to follow a ‘jack-and-master’ strategy, i.e. it is able to maintain high fitness under a wide range of environmental conditions, but performs particularly well in favourable habitats. Therefore, the proposed colonization sequence is likely to be based primarily on changes in propagule pressure. It is concluded that invasive alien plants can become dominant in distinct habitats without local adaptation.     

Chimeric mitochondrial minichromosomes of the human body louse, Pediculus humanus: evidence for homologous and non-homologous recombination

The mitochondrial (mt) genome of the human body louse, Pediculus humanus, consists of 18 minichromosomes. Each minichromosome is 3 to 4 kb long and has 1 to 3 genes. There is unequivocal evidence for recombination between different mt minichromosomes in P. humanus. It is not known, however, how these minichromosomes recombine. Here, we report the discovery of eight chimeric mt minichromosomes in P. humanus. We classify these chimeric mt minichromosomes into two groups: Group I and Group II. Group I chimeric minichromosomes contain parts of two different protein-coding genes that are from different minichromosomes. The two parts of protein-coding genes in each Group I chimeric minichromosome are joined at a microhomologous nucleotide sequence; microhomologous nucleotide sequences are hallmarks of non-homologous recombination. Group II chimeric minichromosomes contain all of the genes and the non-coding regions of two different minichromosomes. The conserved sequence blocks in the non-coding regions of Group II chimeric minichromosomes resemble the "recombination repeats" in the non-coding regions of the mt genomes of higher plants. These repeats are essential to homologous recombination in higher plants. Our analyses of the nucleotide sequences of chimeric mt minichromosomes indicate both homologous and non-homologous recombination between minichromosomes in the mitochondria of the human body louse.     

Aflatoxin B1-2,3-oxide: evidence for its formation in rat liver in vivo and by human liver microsomes in vitro

Injection of [³H]aflatoxin B₁ into rats yielded covalently bound derivatives in hepatic DNA, rRNA, and protein. Mild acid hydrolysis of the DNA and rRNA adducts formed a derivative indistinguishable from 2,3-dihydro-2,3-dihydroxy-aflatoxin B₁. The data indicate that approximately 60% of the nucleic acid adducts were derived from reactions $\text{in vivo}$ with aflatoxin B₁-2,3-oxide. Acid hydrolysis of rRNA-[³Haflatoxin B₁ adduct formed by human liver microsomes $\text{in vitro}$ also liberated the dihydrodiol in significant amount. The 2,3-oxide of aflatoxin B₁ is a probable ultimate carcinogenic metabolite.     

Chromosome-specific subsets of human alpha satellite DNA: Analysis of sequence divergence within and between chromosomal subsets and evidence for an ancestral pentameric repeat

Summary The centromeric regions of human chromosomes are characterized by diverged chromosome-specific subsets of a tandemly repeated DNA family, alpha satellite, which is based on a fundamental monomer repeat unit 171 bp in length. We have compared the nucleotide sequences of 44 alphoid monomers derived from cloned representatives of the multimeric higher-order repeat units of human chromosomes 1, 11, 17, and X. The 44 monomers exhibit an average 16% divergence from a consensus alphoid sequence, and can be assigned to five distinct homology groups based on patterns of sequence substitutions and gaps relative to the consensus. Approximately half of the overall sequence divergence can be accounted for by sequence changes specific to a particular homology group; the remaining divergence appears to be independent of the five groups and is randomly distributed, both within and between chromosomal subsets. The data are consistent with the proposal that the contemporary tandem arrays on chromosomes 1, 11, 17, and X derive from a common multimeric repeat, consisting of one monomer each from the five homology groups. The sequence comparisons suggest that this pentameric repeat must have spread to these four chromosomal locations many millions of years ago, since which time evolution of the four, now chromosome-specific, alpha satellite subsets has been essentially independent.     

Desulfonation of propanesulfonic acid by Comamonas acidovorans strain P53: evidence for an alkanesulfonate sulfonatase and an atypical sulfite dehydrogenase

Evidence is presented for the presence in propanesulfonate-grown Comamonas acidovorans strain P53 of a cytoplasmically located sulfonatase that does not sediment at 100,000 × g. This enzyme catalysed the sulfonate-dependent oxidation of NADH or NADPH, indicating a monooxygenase that effects the addition of molecular oxygen to C₃-C₆ 1-alkanesulfonates. Enzyme activity was proportional to protein concentration only above approximately 2 mg cytoplasmic fraction protein ml^–1, suggesting that the sulfonatase is a multicomponent enzyme, possibly comparable with methanesulfonate monooxygenase. Enzyme activity was strongly inhibited by divalent metal-chelating agents, but was insensitive to cyanide and azide. Sulfite released from sulfonates by Comamonas acidovorans was oxidized by an unusual sulfite dehydrogenase. This was purified approximately 230-fold and was shown to have a molecular mass of 74.4 kDa, comprising two or more subunits. The enzyme activity was specific in vitro for ferricyanide as an electron acceptor and, unlike other bacterial sulfite dehydrogenases, did not contain native cytochrome c or reduce added cytochrome c. It was a basic protein, insensitive to chloride and sulfate, and exhibited a K _m for sulfite of approximately 45 μM. Received: 19 May 1999 / Accepted: 3 September 1999     

Stored mRNA in cotyledons of Vigna unguiculata seeds: nucleotide sequence of cloned cDNA for a stored mRNA and induction of its synthesis by precocious germination

By differential hybridization screening, we previously selected a class of cDNA clones from a gt10 cDNA library that was constructed from the total poly(A)⁺ RNA of mature cowpea cotyledons (Plant Cell Physiol 31: 39–44, 1990). pSAS10, a clone of this class, hybridized with a cDNA probe complementary to poly(A)⁺ RNA from cotyledons collected 1 day after the onset of imbibition (DAI), but not with the cDNA probe from cotyledons at development stage II (13 to 15 days after flowering, DAF). pSAS10 mRNA was detectable only in cotyledons at development stage III (17 to 19 DAF) or later, and its level began to decline when seeds germinated. We have suggested that pSAS10 mRNA is likely to belong to the class of stored mRNA or the mRNA that is formed at the late stage of seed maturation, is conserved in quiescent seeds and becomes functional at the early stage of germination. We determined the nucleotide sequence of pSAS10 cDNA consisting of 459 bp and an approximately 36 bp poly(A) tract, and deduced the amino acid sequence of its product, a 10-kDa cysteine-rich polypeptide. Synthesis of pSAS10 mRNA was induced just before germination began, not only in mature seeds but also in immature seeds even at stages I (9 to 11 DAF) and II (13 to 15 DAF) if they were placed under conditions suitable for germination.     

In silico sequence analysis of arylamine N-acetyltransferases: evidence for an absence of lateral gene transfer from bacteria to vertebrates and first description of paralogs in bacteria

The arylamine N-acetyltransferases (NATs) are xenobiotic-metabolizing enzymes responsible for the biotransformation of various arylamine and heterocyclic amines, including drugs and carcinogenic compounds. NAT and NAT-like genes have been identified in several vertebrate and eubacterial species. Little is known about their evolutionary history, but the horizontal transfer of NAT genes from bacteria to vertebrates was recently suggested [S. Salzberg, O. White, J. Peterson, J. Eisen, Science 292 (2001) 1903]. We used various bioinformatics-based approaches to screen eukaryotic and prokaryotic genomes. We identified Mesorhizobium loti NAT genes as the first examples of NAT paralogs in prokaryotes. As shown for vertebrate species, the existence of NAT paralogs in this bacterium may be accounted for by enzymatic specialization after gene duplication. Phylogenetic analysis following the identification of a NAT ortholog in the nonvertebrate species Ciona intestinalis indicated that NAT genes are unlikely to be examples of direct horizontal gene transfer (HGT). Our study suggests that NAT genes have evolved from a common ancestor, with a succession of nonvertebrate intermediates. The absence of NAT genes in yeast, nematode worms, fruit flies, and mustard weed may result from gene loss in these nonvertebrate lineages. These results provide new insight into the taxonomic distribution and evolutionary history of this class of drug-metabolizing enzymes.     

Degradation of some phenols and hydroxybenzoates by the imperfect ascomycetous yeastsCandida parapsilosis andArxula adeninivorans: evidence for an operative gentisate pathway

The imperfect ascomycetous yeastsCandida parapsilosis andArxula adeninivorans degraded 3-hydroxybenzoic acid via gentisate which was the cleavage substrate. 4-Hydroxybenzoic acid was metabolized via protocatechuate. No cleavage enzyme for the latter was detected. In stead of this NADH- and NADPH-dependent monooxygenases were present. In cells grown at the expense of hydroquinone and 4-hydroxygenzoic acid, enzymes of the hydroxyhydroquinone variant of the 3-oxoadipate pathway were demonstrated, which also took part in the degradation of 2,4-dihydroxybenzoic acid byC. parapsilosis.Abbreviations HHQ Hydroxyhydroquinone (1,2,4-trihydroxybenzene) - GSH reduced Glutathione     

The structural mimicry of membrane sterols by tamoxifen: evidence from cholesterol coefficients and molecular-modelling for its action as a membrane anti-oxidant and an anti-cancer agent

The anti-cancer drug tamoxifen is a potent inhibitor of lipid peroxidation induced by Fe(III)-ascorbate in ox-brain phospholipid liposomes. Similar anti-oxidant effects, but with varying potencies, are also shown by 4-hydroxytamoxifen, cholesterol, ergosterol and 17-β-oestradiol. We now describe a computer-graphic fitting technique that demonstrates a structural similarity between the five compounds. In addition, we have quantified the differences (relative to cholesterol) between the anti-oxidant activities of the compounds in terms of a novel expression reffered to here as the cholesterol coefficient (C_c) Finally, we discuss how the inhibitory effect of tamoxifen on lipid peroxidation may result from a membrane stabilization that is associated with a decrease in membrane fluidity. This action may be related to the anti-proliferative effect exerted by tamoxifen on cancer and fungal cells.     

Formation of an adduct by clenbuterol, a beta-adrenoceptor agonist drug, and serum albumin in human saliva at the acidic pH of the stomach: evidence for an aryl radical-based process

Clenbuterol (CLB) is an antiasthmatic drug used also illegally as a lean muscle mass enhancer in both humans and animals. CLB and amine-related drugs in general are nitrosatable, thus raising concerns regarding possible genotoxic/carcinogenic activity. Oral administration of CLB raises the issue of its possible transformation by salivary nitrite at the acidic pH of gastric juice. In acidic human saliva CLB was rapidly transformed to the CLB arenediazonium ion. This suggests a reaction of CLB with salivary nitrite, as confirmed in aerobic HNO(2) solution by a drastic decrease in nitric oxide, nitrite, and nitrate. In human saliva, both glutathione and ascorbic acid were able to inhibit CLB arenediazonium formation and to react with preformed CLB arenediazonium. The effect of ascorbic acid is particularly pertinent because this vitamin is actively concentrated within the gastric juice. EPR spin trapping experiments showed that preformed CLB arenediazonium ion was reduced to the aryl radical by ascorbic acid, glutathione, and serum albumin, the major protein of saliva. As demonstrated by anti-CLB antibodies and MS, the CLB-albumin interaction leads to the formation of a covalent drug-protein adduct, with a preference for Tyr-rich regions. This study highlights the possible hazards associated with the use/abuse of this drug.     

The complete mitochondrial genome of the Chinese hook snout carp Opsariichthys bidens (Actinopterygii: Cypriniformes) and an alternative pattern of mitogenomic evolution in vertebrate

The complete mitochondrial genome sequence of the Chinese hook snout carp, Opsariichthys bidens, was newly determined using the long and accurate polymerase chain reaction method. The 16,611-nucleotide mitogenome contains 13 protein-coding genes, two rRNA genes (12S, 16S), 22 tRNA genes, and a noncoding control region. We use these data and homologous sequence data from multiple other ostariophysan fishes in a phylogenetic evaluation to test hypothesis pertaining to codon usage pattern of O. bidens mitochondrial protein genes as well as to re-examine the ostariophysan phylogeny. The mitochondrial genome of O. bidens reveals an alternative pattern of vertebrate mitochondrial evolution. For the mitochondrial protein genes of O. bidens, the most frequently used codon generally ends with either A or C, with C preferred over A for most fourfold degenerate codon families; the relative synonymous codon usage of G-ending codons is greatly elevated in all categories. The codon usage pattern of O. bidens mitochondrial protein genes is remarkably different from the general pattern found previously in the relatively closely related zebrafish and most other vertebrate mitochondria. Nucleotide bias at third codon positions is the main cause of codon bias in the mitochondrial protein genes of O. bidens, as it is biased particularly in favor of C over A. Bayesian analysis of 12 concatenated mitochondrial protein sequences for O. bidens and 46 other teleostean taxa supports the monophyly of Cypriniformes and Otophysi and results in a robust estimate of the otophysan phylogeny.