The many ways to delimit species: hairs, genes and surface chemistry

Species identification forms the basis for understanding the diversity of the living world, but it is also a prerequisite for understanding many evolutionary patterns and processes. The most promising approach for correctly delimiting and identifying species is to integrate many types of information in the same study. Our aim was to test how cuticular hydro- carbons, traditional morphometrics, genetic polymorphisms in nuclear markers (allozymes and DNA microsatellites) and DNA barcoding (partial mitochondrial COI gene) perform in delimiting species. As an example, we used two closely related Formica ants, F. fusca and F. lemani, sampled from a sympatric population in the northern part of their distribu- tion. Morphological characters vary and overlap in different parts of their distribution areas, but cuticular hydrocarbons include a strong taxonomic signal and our aim is to test the degree to which morphological and genetic data correspond to the chemical data. In the morphological analysis, species were best separated by the combined number of hairs on pro- notum and mesonotum, but individual workers overlapped in hair numbers, as previously noted by several authors. Nests of the two species were separated but not clustered according to species in a Principal Component Analysis made on nuclear genetic data. However, model-based Bayesian clustering resulted in perfect separation of the species and gave no indication of hybridization. Furthermore, F. lemani and F. fusca did not share any mitochondrial haplotypes, and the species were perfectly separated in a phylogenetic tree. We conclude that F. fusca and F. lemani are valid species that can be separated in our study area relatively well with all methods employed. However, the unusually small genetic differen- tiation in nuclear markers (FST = 0.12) shows that they are closely related, and occasional hybridization between F. fusca and F. lemani cannot be ruled out.

Identification of Mxr1p-binding sites in the promoters of genes encoding dihydroxyacetone synthase and peroxin 8 of the methylotrophic yeast Pichia pastoris

Expression of genes involved in methanol metabolism of Pichia pastoris is regulated by Mxr1p, a zinc finger transcription factor. In this study, we studied the target gene specificity of Mxr1p by examining its ability to bind to promoters of genes encoding dihydroxyacetone synthase (DHAS) and peroxin 8 (PEX8), since methanol-inducible expression of these genes is abrogated in mxr1-null mutant strains of P. pastoris. Different regions of DHAS and PEX8 promoter were isolated from P. pastoris genomic DNA and their ability to bind to a recombinant Mxr1p protein containing the N-terminal 150 amino acids, including the zinc finger DNA-binding domain, was examined. These studies reveal that Mxr1p specifically binds to promoter regions containing multiple 5'-CYCC-3' sequences, although all DNA sequences containing the 5'-CYCC-3' motif do not qualify as Mxr1p-binding sites. Key DNA-binding determinants are present outside 5'-CYCC-3' motif and Mxr1p preferably binds to DNA sequences containing 5'-CYCCNY-3' than those containing 5'-CYCCNR-3' sequences. This study provides new insights into the molecular determinants of target gene specificity of Mxr1p, and the methodology described here can be used for mapping Mxr1p-binding sites in other methanol-inducible promoters of P. pastoris. Copyright (C) 2010 John Wiley & Sons, Ltd.

Crucial contribution of the multiple copies of the initiator tRNA genes in the fidelity of tRNA(fMet) selection on the ribosomal P-site in Escherichia coli

The accuracy of the initiator tRNA (tRNA(fMet)) selection in the ribosomal P-site is central to the fidelity of protein synthesis. A highly conserved occurrence of three consecutive G-C base pairs in the anticodon stem of tRNA(fMet) contributes to its preferential selection in the P-site. In a genetic screen, using a plasmid borne copy of an inactive tRNA(fMet) mutant wherein the three G-C base pairs were changed, we isolated Escherichia coli strains that allow efficient initiation with the tRNA(fMet) mutant. Here, extensive characterization of two such strains revealed novel mutations in the metZWV promoter severely compromising tRNA(fMet) levels. Low cellular abundance of the chromosomally encoded tRNA(fMet) allows efficient initiation with the tRNA(fMet) mutant and an elongator tRNA(Gln), revealing that a high abundance of the cellular tRNA(fMet) is crucial for the fidelity of initiator tRNA selection on the ribosomal P-site in E. coli. We discuss possible implications of the changes in the cellular tRNA(fMet) abundance in proteome remodeling.

Cloning and characterization of a cluster of genes coding for 5S rRNA and three tRNAs from Azospirillum lipoferum

A genomic library was constructed from a HindIII digest of Azospirillum lipoferum chromosomal DNA in the HindIII site of pUC19. From the library, a clone, pALH64, which showed strong hybridization with 3' end labeled A. lipoferum total tRNAs and which contains a 2.9 kb insert was isolated and restriction map of the insert established. The nucleotide sequence of a 490 bp HindIII-HincII subfragment containing a cluster of genes coding for 5S rRNA, tRNA(Val)(UAC), tRNA(Thr)(UGA) and tRNA(Lys)(UUU) has been determined. The gene organization is 5S rRNA (115 bp), spacer (10 bp), tRNA(Val) (76 bp), spacer (3 bp), tRNA(Thr) (76 bp), spacer (7 bp) and tRNA(Lys) (76 bp). Hybridization experiments using A. lipoferum total tRNAs and 5S rRNA with the cloned DNA probes revealed that all three tRNA genes and the 5S rRNA gene are expressed in vivo in the bacterial cells.

Conformationally Restricted Criegee Intermediates: Evidence for Formation and Stereoelectronically Controlled Fragmentation

The Baeyer-Villiger reaction of 2-(2-oxocyclohexyl) acetic acid occurs via a bicyclic Criegee intermediate, which fragments with stereoelectronic control, as evidenced by product analysis; the reaction of the but-2-yl ester and of 2-(2-oxocyclopentyl) acetic acid also show evidence of such stereoelectronic control, but less convincingly.

Identification and characterization of a new plasmid carrying genes for degradation of 2,4-dichlorophenoxyacetate from Pseudomonas cepacia CSV90

Pseudomonas cepacia CSV90 is able to utilize 2,4-dichlorophenoxyacetate (2,4-D) and 2-methyl-4-chlorophenoxyacetate as sole sources of carbon and energy. Mutants of the strain CSV90 which had lost this ability appeared spontaneously on a nonselective medium. The wild-type strain harbored a 90-kb plasmid, pMAB1, whereas 2,4-D-negative mutants either lost the plasmid or had a 70-kb plasmid, pMAB2. The plasmid pMAB2 was found to have undergone a deletion Of a 20-kb fragment of pMAB1. The plasmid-free mutants regained the ability to degrade 2,4-D after introduction of purified pMAB1 by electroporation. Cloning in Escherichia coli of a 10-kb BamHI fragment from pMAB1, the region absent in pMAB2, resulted in the expression of the gene tfdC encoding 3,5-dichlorocatechol 1,2-dioxygenase. After subcloning, the tfdC gene was located in a 1.6-kb HindIII fragment. The nucleotide sequence of the tfdC gene and the restriction map of its contiguous region are identical to those of the well-characterized 2,4-D-degradative plasmid pJP4 of Alcaligenes eutrophus, whereas the overall restriction maps of the two plasmids are different. The N-terminal 44-amino-acid sequence of the enzyme purified from the strain CSV90 confirmed the reading frame in the DNA sequence for tfdC and indicated that the initiation codon GUG is read as methionine instead of valine.

Adaptive optimal tuning of a general class of stable LTI systems with restricted inputs

The problem addressed is one of model reference adaptive control (MRAC) of asymptotically stable plants of unknown order with zeros located anywhere in the s-plane except at the origin. The reference model is also asymptotically stable and lacking zero(s) at s = 0. The control law is to be specified only in terms of the inputs to and outputs of the plant and the reference model. For inputs from a class of functions that approach a non-zero constant, the problem is formulated in an optimal control framework. By successive refinements of the sub-optimal laws proposed here, two schemes are finally design-ed. These schemes are characterized by boundedness, convergence and optimality. Simplicity and total time-domain implementation are the additional striking features. Simulations to demonstrate the efficacy of the control schemes are presented.

Detection of nif genes in nitrogen-fixing bacterial isolate from tomato (Lycopersicon esculentum Mill cv Pusa ruby)

Nitrogen-fixing bacterial isolate from the intercellular spaces of tomato root cortical cells was studied for the location of nif genes on the chromosomal or plasmid DNA. The bacterial isolate showed two plasmids of approximate molecular sizes of 220 and 120 kb. Klebsiella pneumoniae nif HDK probe hybridized with the chromosomal DNA and not with the plasmid DNA thereby showing that nif genes are localised on the chromosomal DNA.

Mismatch repair genes of eukaryotes

Mismatches that arise during replication or genetic recombination or owing to damage to DNA by chemical agents are recognized by mismatch repair systems. The pathway has been characterized in detail in Escherichia coli. Several homologues of the genes encoding the proteins of this pathway have been identified in the yeast Saccharomyces cerevisiae and in human cells. Mutations in the human genes hMSH2, hMLH1, hPMS1 and hPMS2 have been linked to hereditary nonpolyposis colon cancer (HNPCC) and to some sporadic tumours. Mismatch repair also plays an antirecombinogenic role and is implicated in speciation.

Ultrafast Solvation Dynamics Of An Ion In A Restricted Environment

The solvation dynamics of an excited coumarin dye molecule (C-480) enclosed within a restricted space have been studied using molecular hydrodynamic theory (MHT) and compared with the recent experimental findings. The solvation dynamics of the dye molecule within the cavity of a toroidal gamma-cyclodextrin molecule have been shown to be explained only in terms of the freezing of the solvent translational modes using MHT. The results of the theoretical calculation are in good agreement with the experimental results. The inertial components of the solvation time correlation function remain the same in both the restricted environment and in the free space. These results are interesting in the light of the simulation studies of Maroncelli and Fleming [J chem Phys, 89 (1988) 5044] which concludes that the participation of the different solvation shells in controlling the dynamics are much different. The earlier studies have been reviewed and the recent findings are discussed.

Design and structural analysis of hairpin-TFO for transcriptional activation of genes in S-cerevisiae

Triplex forming oligonucleotides (TFOs) have the potential to modulate gene expression. While most of the experiments are directed towards triplex mediated inhibition of gene expression the strategy potentially could be used for gene specific activation. In an attempt to design a strategy for gene specific activation in vivo applicable to a large number of genes we have designed a TFO based activator-target system which may be utilized in Saccharomyces cerevisiae or any other system where Gal4 protein is ectopically expressed. The total genome sequence of Saccharomyces cerevisiae and expression profiles were used to select the target genes with upstream poly (pu/py) sequences. We have utilized the paradigm of Gal4 protein and its binding site. We describe here the selection of target genes and design of hairpin-TFO including the targeting sequences containing polypurine stretch found in the upstream promoter regions of weakly expressed genes. We demonstrate, the formation of hairpin-TFO, its binding to Gal4 protein, its ability to form triplex with the target duplex in vitro, the effect of polyethylenimine on complex formation and discuss the implication on in vivo transcription activation.

Expression pattern of Cubitus interruptus from the mulberry silkworm Bombyx mori in late developmental stages

A partial genomic clone of Bombyx mori homologue of the segment polarity gene Cubitus interruptus (BmCi), encoding the conserved zinc finger domain and harbouring two introns, has been characterized. BmCi was expressed in the silkglands of B. mori from embryonic to the late larval stages(3rd, 4th and 5th intermoults). The expression was confined to the anterior region of the middle silkglands, overlapping with the domain of sericin-2 expression and excluding the domains of Bm invected expression, namely the middle and posterior regions of the middle silkglands. In the wing discs, the expression was restricted to the anterior compartment, which increased from 4th to 5th larval intermoults and declined later in the pupal wing buds. In gonadal tissues (both ovaries and testes) BmCi was expressed from the larval to pupal stages. The transcripts were localized to the sperm tubes containing spermatogonia in the testis of Bombyx larvae. BmCi expression, however, was not detected in any of these tissues during the moulting stages. Expression of Ci in the wing discs and gonads is evolutionarily conserved, while the silkgland represents a novel domain. Our results imply that BmCi is involved in the specification and maintenance of micro-compartment identity within the middle silkglands.

The beta-glucoside genes of Klebsiella aerogenes: conservation and divergence in relation to the cryptic bgl genes of Escherichia coli

The ability to metabolize aromatic beta-glucosides such as salicin and arbutin varies among members of the Enterobacteriaceae. The ability of Escherichia coli to degrade salicin and arbutin appears to be cryptic, subject to activation of the bgl genes, whereas many members of the Klebsiella genus can metabolize these sugars. We have examined the genetic basis for beta-glucoside utilization in Klebsiella aerogenes. The Klebsiella equivalents of bglG, bglB and bglR have been cloned using the genome sequence database of Klebsiella pneumoniae. Nucleotide sequencing shows that the K. aerogenes bgl genes show substantial similarities to the E. coli counterparts. The K. aerogenes bgl genes in multiple copies can also complement E. coli mutants deficient in bglG encoding the antiterminator and bglB encoding the phospho-beta-glucosidase, suggesting that they are functional homologues. The regulatory region bglR of K aerogenes shows a high degree of similarity of the sequences involved in BglG-mediated regulation. Interestingly, the regions corresponding to the negative elements present in the E. coli regulatory region show substantial divergence in K aerogenes. The possible evolutionary implications of the results are discussed. (C) 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.v. All rights reserved.

Features of a unique intronless cluster of class I small heat shock protein genes in tandem with box C/D snoRNA genes on chromosome 6 in tomato (Solanum lycopersicum)

Physical clustering of genes has been shown in plants; however, little is known about gene clusters that have different functions, particularly those expressed in the tomato fruit. A class I 17.6 small heat shock protein (Sl17.6 shsp) gene was cloned and used as a probe to screen a tomato (Solanum lycopersicum) genomic library. An 8.3-kb genomic fragment was isolated and its DNA sequence determined. Analysis of the genomic fragment identified intronless open reading frames of three class I shsp genes (Sl17.6, Sl20.0, and Sl20.1), the Sl17.6 gene flanked by Sl20.1 and Sl20.0, with complete 5' and 3' UTRs. Upstream of the Sl20.0 shsp, and within the shsp gene cluster, resides a box C/D snoRNA cluster made of SlsnoR12.1 and SlU24a. Characteristic C and D, and C' and D', boxes are conserved in SlsnoR12.1 and SlU24a while the upstream flanking region of SlsnoR12.1 carries TATA box 1, homol-E and homol-D box-like cis sequences, TM6 promoter, and an uncharacterized tomato EST. Molecular phylogenetic analysis revealed that this particular arrangement of shsps is conserved in tomato genome but is distinct from other species. The intronless genomic sequence is decorated with cis elements previously shown to be responsive to cues from plant hormones, dehydration, cold, heat, and MYC/MYB and WRKY71 transcription factors. Chromosomal mapping localized the tomato genomic sequence on the short arm of chromosome 6 in the introgression line (IL) 6-3. Quantitative polymerase chain reaction analysis of gene cluster members revealed differential expression during ripening of tomato fruit, and relatively different abundances in other plant parts.