A conserved sequence in tRNA and rRNA promoters of Lactococcus lactis

A tRNA operon (trnA) from Lactococcus lactis consisting of seven tRNA genes and a 5S rRNA gene was cloned and sequenced. Promoter-fusion of the trnA promoter to a promoter-less beta-galactosidase gene of Leuconostoc mesenteroides resulted in high levels of beta-galactosidase activity in L. lactis. Searching for sequences with similarity to the sequence of the promoter region revealed a consensus sequence of promoters preceeding rRNA operons and tRNA operons from Lactococcus species including a not previously described conserved sequence (AGTT).     

tRNA genes transcribed from the plastid-like DNA of Plasmodium falciparum

Besides their mitochondrial genome, malarial parasites contain a second organellar DNA. This 35 kb circular molecule has a number of features reminiscent of plastid DNAs. Sequence analysis shows that along with other genes the circle codes for 25 different tRNAs all of which are transcribed. Six of the tRNAs have some unusual features, and one has an intron, the only one found so far on the circle. Comparison of codon and anticodon usage indicates that the 25 tRNAs are sufficient to decode all the protein genes present on the circle. The maintenance of such a parsimonious but complete translation system is further evidence for the functionality of the circle.     

Effects of exogenous phytohormones on plastid tRNA modifications in ragi coleoptiles

The influence of adenosine and selective A1 and A2 agonists and antagonists was investigated on the cholinergic and the excitatory non-cholinergic (e-NC) contractions induced by electrical field stimulation in the guinea-pig bronchi. Adenosine (10 nM-1 mM) induced a concentration-dependent inhibition of the e-NC contraction (EC50 = 90 +/- 14 microM), whereas the cholinergic peak was only slightly affected. Preincubation of the tissue with the adenosine uptake blocker dipyridamole (10 microM) significantly shifted the concentration-inhibition curve to adenosine to the left (EC50 = 10 +/- 1 microM), suggesting an interaction with extracellular adenosine receptors of A1 and/or A2 subtype. To characterize the receptor type involved in this effect, selective adenosine derivatives were studied. The agonist to both A1 and A2 adenosine receptors, 5'-N-ethylcarboxamidoadenosine (NECA) was more potent than the selective A1 agonist, (-)-R-6-phenylisopropyladenosine (R-PIA), in inhibiting the e-NC contraction (EC50 = 0.10 +/- 0.04 and 0.60 +/- 0.12 microM, respectively, with a maximal inhibition of 70 and 45%, respectively). The concentration-response curve to NECA was shifted to the right by the A2 receptor selective antagonist 3,7-dimethyl-1-propargylxanthine (DMPX) (10 microM) (EC50 = 1.4 +/- 0.5 microM) as well as by the specific A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) (10 microM) (EC50 = 0.7 +/- 0.3 microM). The inhibitory effect induced by the association of both antagonists, DPCPX and DMPX, was considerably potentiated (EC50 > 22 +/- 2.5 microM). The effect of R-PIA was also shifted to the right by DPCPX (EC50 = 8.2 +/- 1.6 microM) but was not modified by DMPX. The contractile response to exogenous substance P was unaffected by NECA pretreatment (0.3 microM). Altogether, these results suggest that adenosine-induced inhibition of e-NC contraction of guinea-pig bronchi is mediated through activation of both A1 and A2 adenosine receptors linked to inhibition of the release of neuropeptides from C-fibre nerve endings.     

Two 16S-23S ribosomal DNA intergenic regions in different Treponema pallidum subspecies contain tRNA genes

The 16S-23S intergenic spacers of Treponema pallidum subspecies pallidum, Nichols strain, and Treponema pallidum subspecies pertenue, Gauthier strain, have been cloned, characterized and sequenced. Isoleucine and alanine tRNA genes have been identified within the 16S-23S intergenic regions on separate alleles of 293 and 303 bases, respectively. The two alleles are present in both T.p.pallidum and T.p. pertenue, and show no sequence differences between the bacterial subspecies. The ile-tRNA and ala-tRNA genes show 65% and 84% sequence identity, respectively, with the homologous genes of the related spirochete, Borrelia burgdorferi.     

A functional homolog of a yeast tRNA splicing enzyme is conserved in higher eukaryotes and in Escherichia coli

tRNA splicing in the yeast Saccharomyces cerevisiae requires an endonuclease to excise the intron, tRNA ligase to join the tRNA half-molecules, and 2'-phosphotransferase to transfer the splice junction 2'-phosphate from ligated tRNA to NAD, producing ADP ribose 1"-2" cyclic phosphate (Appr>p). We show here that functional 2'-phosphotransferases are found throughout eukaryotes, occurring in two widely divergent yeasts (Candida albicans and Schizosaccharomyces pombe), a plant (Arabidopsis thaliana), and mammals (Mus musculus); this finding is consistent with a role for the enzyme, acting in concert with ligase, to splice tRNA or other RNA molecules. Surprisingly, functional 2'-phosphotransferase is found also in the bacterium Escherichia coli, which does not have any known introns of this class, and does not appear to have a ligase that generates junctions with a 2'-phosphate. Analysis of the database shows that likely members of the 2'-phosphotransferase family are found also in one other bacterium (Pseudomonas aeruginosa) and two archaeal species (Archaeoglobus fulgidus and Pyrococcus horikoshii). Phylogenetic analysis reveals no evidence for recent horizontal transfer of the 2'-phosphotransferase into Eubacteria, suggesting that the 2'-phosphotransferase has been present there since close to the time that the three kingdoms diverged. Although 2'-phosphotransferase is not present in all Eubacteria, and a gene disruption experiment demonstrates that the protein is not essential in E. coli, the continued presence of 2'-phosphotransferase in Eubacteria over large evolutionary times argues for an important role for the protein.     

A conserved aspartate of tRNA pseudouridine synthase is essential for activity and a probable nucleophilic catalyst

tRNA pseudouridine synthase I catalyzes the conversion of uridine to pseudouridine at positions 38, 39, and/or 40 in the anticodon loop of many tRNAs. Pseudouridine synthase I was cloned behind a T7 promoter and expressed in Escherichia coli to about 20% of total soluble proteins. Fluorouracil-substituted tRNA caused a time-dependent inactivation of pseudouridine synthase I and formed a covalent complex with the enzyme that involved the FUMP at position 39. Asp60, conserved in all known and putative pseudouridine synthases, was mutated to amino acids with diverse side chains. All Asp60 mutants bound tRNA but were catalytically inactive and failed to form covalent complexes with fluorouracil-substituted tRNA. We conclude that the conserved Asp60 is essential for pseudouridine synthase activity and propose mechanisms which involve this residue in important catalytic roles.     

DNA repair in higher plants

Numerous studies have demonstrated a requirement in plants for repair of DNA damage arising from either intrinsic or extrinsic sources. Investigations also have revealed a capacity for repair of certain types of DNA damage, and conversely, identified mutants apparently defective in such repair. This article provides a concise overview of nuclear DNA repair mechanisms in higher plants, particularly those processes concerned with the repair of UV-induced lesions, and includes surveys of UV-sensitive mutants and genes implicated in DNA repair.     

Identification of conserved, RpoS-dependent stationary-phase genes of Escherichia coli

During entry into stationary phase, many free-living, gram-negative bacteria express genes that impart cellular resistance to environmental stresses, such as oxidative stress and osmotic stress. Many genes that are required for stationary-phase adaptation are controlled by RpoS, a conserved alternative sigma factor, whose expression is, in turn, controlled by many factors. To better understand the numbers and types of genes dependent upon RpoS, we employed a genetic screen to isolate more than 100 independent RpoS-dependent gene fusions from a bank of several thousand mutants harboring random, independent promoter-lacZ operon fusion mutations. Dependence on RpoS varied from 2-fold to over 100-fold. The expression of all fusion mutations was normal in an rpoS/rpoS+ merodiploid (rpoS background transformed with an rpoS-containing plasmid). Surprisingly, the expression of many RpoS-dependent genes was growth phase dependent, albeit at lower levels, even in an rpoS background, suggesting that other growth-phase-dependent regulatory mechanisms, in addition to RpoS, may control postexponential gene expression. These results are consistent with the idea that many growth-phase-regulated functions in Escherichia coli do not require RpoS for expression. The identities of the 10 most highly RpoS-dependent fusions identified in this study were determined by DNA sequence analysis. Three of the mutations mapped to otsA, katE, ecnB, and osmY-genes that have been previously shown by others to be highly RpoS dependent. The six remaining highly-RpoS-dependent fusion mutations were located in other genes, namely, gabP, yhiUV, o371, o381, f186, and o215.     

Reconstruction of quaternary structures of class II tRNA synthetases by rational mutagenensis of a conserved domain

Class II tRNA synthetases have long been known to have quaternary structures of alpha, alpha2, alpha2beta2, and alpha4, depending on the amino acid specificity and the organism from which the synthetase was isolated. Even the quaternary structures of enzymes for the same amino acid show variations in evolution. The basis for these variations has not been understood. We report here that sequence manipulations of a structural motif (motif 1) characteristic of all class II tRNA synthetases can generate most of the evolutionary diversity of quaternary forms of class II synthetases. Thus, the principles elucidated here for quaternary structure assembly may be general.     

Molecular mapping and tagging of genes in crop plants

In India, molecular mapping and tagging of agronomically important genes using RFLP and RAPD markers have been carried out in three different crops: rice, mustard and chickpea. In rice, tagging of genes for resistance to gall midge and blast has been accomplished. Molecular mapping of cooking quality traits in rice is in progress. For fingerprinting rice cultivars, suitable probe enzyme combinations have been identified. In mustard, a partial RFLP linkage map has been constructed and one of the yellow seed-coat colour loci has been mapped. Significant associations of RFLP markers with quantitative traits have also been established. Potential use of RAPD markers to identify heterotic groups among mustard accessions has been demonstrated. In chickpea, the occurrence of considerable interspecific DNA polymorphism as revealed by RAPD analysis has facilitated construction of a partial linkage map.     

A conserved mode of head segmentation in arthropods revealed by the expression pattern of Hox genes in a spider

Chelicerates constitute a basic arthropod group with fossil representatives from as early as the Cambrian period. Embryonic development and the subdivision of the segmented body region into a prosoma and an opisthosoma are very similar in all extant chelicerates. The mode of head segmentation, however, has long been controversial. Although all other arthropod groups show a subdivision of the head region into six segments, the chelicerates are thought to have the first antennal segment missing. To examine this problem on a molecular level, we have compared the expression pattern of Hox genes in the spider Cupiennius salei with the pattern known from insects. Surprisingly, we find that the anterior expression borders of the Hox genes are in the same register and the same relative segmental position as in Drosophila. This contradicts the view that the homologue of the first antennal segment is absent in the spider. Instead, our data suggest that the cheliceral segment is homologous to the first antennal segment and the pedipalpal segment is homologous to the second antennal (or intercalary) segment in arthropods. Our finding implies that chelicerates, myriapods, crustaceans, and insects share a single mode of head segmentation, reinforcing the argument for a monophyletic origin of the arthropods.     

A novel class of evolutionarily conserved genes defined by piwi are essential for stem cell self-renewal

Germ-line stem cells (GSCs) serve as the source for gametogenesis in diverse organisms. We cloned and characterized the Drosophila piwi gene and showed that it is required for the asymmetric division of GSCs to produce and maintain a daughter GSC but is not essential for the further differentiation of the committed daughter cell. Genetic mosaic and RNA in situ analyses suggest that piwi expression in adjacent somatic cells regulates GSC division. piwi encodes a highly basic novel protein, well conserved during evolution. We isolated piwi homologs in Caenorhabditis elegans and humans and also identified Arabidopsis piwi-like genes known to be required for meristem cell maintenance. Decreasing C. elegans piwi expression reduces the proliferation of GSC-equivalent cells. Thus, piwi represents a novel class of genes required for GSC division in diverse organisms.     

Finding the genes in genomic DNA

We analyzed two-locus disequilibria for 16 polymorphic loci of seven susceptibility genes for coronary artery disease located in five chromosomal regions distributed across four chromosomes. Included were the genes coding for apolipoprotein B (ApoB, chromosome 2, four marker loci), lipoprotein lipase (LPL, chromosome 8, three marker loci), apolipoproteins AI, CIII AIV (ApoAI-CIII-AIV, chromosome 11, three marker loci), apolipoprotein E (ApoE, chromosome 19, two marker loci), and the low density lipoprotein receptor (LDLR, chromosome 19, four marker loci). Our sample included 540 unrelated individuals from the Rochester, Minn. population. There were no statistically significant deviations of single-locus genotypes from Hardy-Weinberg equilibrium. The strongest associations within genes were for composite diallelic disequilibria; 17/19 were significant (13 at Pr <0.001, 1 at Pr <0.01, 3 at Pr <0.05). These observations suggest marker alleles within genes have a shared evolutionary history reflected by disequilibria that have not been dissipated by recombination. Disequilibrium was not generally concordant with the physical orderings of markers. Only two significant higher-order disequilibria were observed although 12 triallelic disequilibria were at maximum possible values. We observed 19 statistically significant disequilibria (Pr <0.05; 4 composite diallelic, 13 triallelic, and 2 quadriallelic) between 101 pairs of marker loci, where each locus in a pair was from a different unlinked region. These unexpected results are most likely explained by recent historical factors, including worldwide population expansion and amalgamation with continuous admixture, that influence the genetic structure (organization of alleles and non-alleles into genotypes) of a population. We conclude that disequilibria between loci from unlinked regions may be more extensive than is commonly assumed. Our findings also suggest that it is, on average, at least 15 times more likely to not detect significant disequilibrium among unlinked loci when it is really present than to make a false positive inference. Disequilibria between functional loci within or between regions will impact estimates of genetic variance associated with particular functional mutations within a susceptibility gene region.     

Polypyrimidine sequences found in eukaryotic DNA have been conserved during evolution

L-cell DNA contains an unexpectedly large amount of long pyrimidine tracts. Hydroxyapatite chromatography has been employed to show that these polypyrimidines hydridize extensively to the reiterated DNA of a large number of eukaryotes but fail to hybridize to prokaryotic DNA. This reaction is sequence specific and not the result of a special property of polypyrimidines since random 3H-labelled poly(dC-dT) shows poor hybridization to eukaryotic DNA. The hybrids formed by L-cell polypyrimidines and heterologous repeated sequences have a higher thermal stability than the corresponding hybrids of total repeated DNA indicating that sequences related to these polypyrimidines have been conserved during evolution. Furthermore, at least some of these tracts are transcribed because they are capable of reacting extensively with total cellular RNA. Although the function of these sequences is not yet known, the fact that they are widely conserved in evolution and also transcribed leads us to speculate that they play an an important role in eukaryotic cells.     

Identification of the conserved coding sequences of three chitin synthase genes in Fonsecaea pedrosoi

Primers having designs based on highly conserved stretches in the deduced amino acid sequences of chitin synthase (CHS) genes were used in PCR reactions to amplify 600 bp and 366 bp products from the genomic DNA of three major causal agents of chromoblastomycosis. Cloning and sequencing of the PCR products of one of these fungi, Fonsecaea pedrosoi, identified three CHS sequences designated as FpCHS1, FpCHS2 and FpCHS3. FpCHS1 and FpCHS2 were homologous to regions of CHS1 and CHS2 of Saccharomyces cerevisiae, and their derived amino acid sequences fell into chitin synthase classes I and II, respectively. FpCHS3 was homologous to a region of the CAL1/CSD2 gene of S. cerevisiae, which codes for the chitin synthase three (Chs3) enzyme in that fungus. Phylogenetic trees constructed using the deduced amino acid sequences of PCR-amplified CHS products from many fungi clustered F. pedrosoi with other dematiaceous fungi, providing new molecular evidence for the genetic relatedness of these organisms. The identification of these CHS genes in F. pedrosoi will facilitate future studies of the functional roles of chitin synthases in the unique in vivo dimorphism exhibited by chromoblastomycotic fungi.     

Analysis of genes encoding highly conserved lysine-rich proteins in Aplysia californica and Saccharomyces cerevisiae

To isolate a gene that can be used as an internal control in studies on gene expression in Aplysia californica neurons, we have characterized a cDNA clone (pKRP-A) isolated on the basis of its high expression in A. californica neurons. This cDNA is of 850 nucleotides and codes for a putative 29-kDa lysine-rich protein. Blotting experiments revealed that the gene is expressed in all tested A. californica tissues, and in individually identified neurons of the abdominal ganglion, suggesting that this gene can be efficiently used as internal control in studies of gene expression. We have also isolated one cDNA and two different genomic clones from yeast libraries that show 59% identity with pKRP-A. Sequence comparison of genomic clones, as well as PCR and Southern blotting experiments, revealed that at least two homologous genes are present in yeast. Northern blotting experiments revealed that the expression of the gene is strongly repressed at 39 degrees C.