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PubMed1999

Limitations to tobacco mosaic virus infection of turnip.

Turnip vein-clearing virus (TVCV) and tobacco mosaic virus (TMV) represent subgroups of tobamoviruses infecting cruciferous and solanaceous plants, respectively. To identify adaptations that may have been necessary in the evolution of the TVCV subgroup from a TMV-like ancestor, the infection of turnip plants by TMV and by chimeras between TMV and TVCV was explored. TMV accumulated at spatially limited sites on inoculated turnip leaves as determined by leaf skeleton hybridization. A plasmid DNA containing a complete TVCV cDNA, when transcribed in vitro, produced RNA that was infectious to tobacco and turnip plants. TVCV-TMV chimeric genomes with junctions within coding regions were not infectious to tobacco, though the movement protein (MP) chimera was infectious to tobacco with a TMV MP transgene. Reciprocal chimeras with junctions between genes were infectious to tobacco. TVCV with a TMV MP gene infected turnips. The other tested chimeras were not detected in non-inoculated leaves, but were found in the inoculated leaves. Thus, the TMV MP is not responsible for the limitation of TMV spread in turnips.

Brassica↗
PubMed2003

A novel RNA splicing-mediated gene silencing mechanism potential for genome evolution.

Over 90% of the human genome consists of non-protein-coding regions. Introns constitute most of the non-coding regions located in precursor messenger RNAs (pre-mRNAs). During pre-mRNA maturation, the introns are excised out of mRNA and thought to be completely digested prior to translation. If the introns were merely metabolic "leavings," why would the genome hold such a large amount of extraneous genetic materials? Here we show a novel posttranscriptional gene silencing system identified within mammalian introns. By packaging human spliceosome-recognition sites along with an exonic insert into an artificial intron, we observed that the splicing and processing of such an exon-containing intron in either sense or antisense conformation produced equivalent gene silencing effects, while a palindromic hairpin insert containing both sense and antisense strands resulted in synergistic effects. These findings may explain how cells respond to the presence of transgenic introns that are homologous to pre-existing exons during genomic evolution.

Animals↗
PubMed2005

A snapshot of viral evolution from genome analysis of the tectiviridae family.

The origin, evolution and relationships of viruses are all fascinating topics. Current thinking in these areas is strongly influenced by the tailed double-stranded (ds) DNA bacteriophages. These viruses have mosaic genomes produced by genetic exchange and so new natural isolates are quite dissimilar to each other, and to laboratory strains. Consequently, they are not amenable to study by current tools for phylogenetic analysis. Less attention has been paid to the Tectiviridae family, which embraces icosahedral dsDNA bacterial viruses with an internal lipid membrane. It includes viruses, such as PRD1, that infect Gram-negative bacteria, as well as viruses like Bam35 with Gram-positive hosts. Although PRD1 and Bam35 have closely related virion morphology and genome organization, they have no detectable sequence similarity. There is strong evidence that the Bam35 coat protein has the "double-barrel trimer" arrangement of PRD1 that was first observed in adenovirus and is predicted to occur in other viruses with large facets. It is very likely that a single ancestral virus gave rise to this very large group of viruses. The unprecedented degree of conservation recently observed for two Bam35-like tectiviruses made it important to investigate those infecting Gram-negative bacteria. The DNA sequences for six PRD1-like isolates (PRD1, PR3, PR4, PR5, L17, PR772) have now been determined. Remarkably, these bacteriophages, isolated at distinctly different dates and global locations, have almost identical genomes. The discovery of almost invariant genomes for the two main Tectiviridae groups contrasts sharply with the situation in the tailed dsDNA bacteriophages. Notably, it permits a sequence analysis of the isolates revealing that the tectiviral proteins can be dissected into a slowly evolving group descended from the ancestor, the viral self, and a more rapidly changing group reflecting interactions with the host.

Amino Acid Sequence↗
PubMed1981

Multiple differences between the nucleic acid sequences of the IgG2aa and IgG2ab alleles of the mouse.

To compare the structure of IgG2a alleles we have determined the complete DNA sequence of the constant region, coding sequence, and 3' untranslated region of a cDNA clone, pAB gamma 2a-1, which was derived from the C57BL/6 mouse strain (b allotype). This sequence was compared with the corresponding IgG2a DNA sequence of BALB/c origin (a allotype). The DNA sequences showed 10% differences, and the deduced protein sequences differed by about 15%. These differences were not evenly distributed: most differences were in the hinge region, the CH3 domain and the 3' untranslated region. It is evident that many alterations in the IgG2a alleles have occurred since the a and b haplotypes were separated--some of these changes were point mutations but some appear to have resulted from gene conversion of the IgG2ab allele by the IgG2bb allele.

Alleles↗
PubMed1985

Structural organization of the DR subregion of the human major histocompatibility complex.

Two clusters of overlapping cosmid and lambda phage clones comprising 205 kilobases (kb) have been isolated from the DR subregion of the human major histocompatibility complex from a DR4 haplotype. A single DR alpha and three DR beta genes were identified. In one cluster (135 kb), the DR alpha gene is 90 kb distant from the DR beta gene encoding a molecule that carries the MT3 serological specificity. In the second cluster (70 kb), the DR beta gene determining the DR4 specificity is located 22 kb apart from a DR beta pseudogene (DR beta psi). A 3- to 4-kb sequence located at the 5' end of the DR beta (MT3) gene is common to all three DR beta-chain genes. In addition, three more copies of this sequence are spaced between the DR alpha and the DR beta (MT3) genes in the first cluster and one of these, at least, is associated with a DR beta 1 exon, suggesting that additional genes could be encoded in this region and that multiple duplication events have led to its evolution.

Biological Evolution↗
PubMed2003

Constructing a feedback loop with circadian clock molecules from the silkmoth, Antheraea pernyi.

Circadian clocks are important regulators of behavior and physiology. The circadian clock of Drosophila depends on an autoinhibitory feedback loop involving dCLOCK, CYCLE (also called dBMAL, for Drosophila brain and muscle ARNT-like protein), dPERIOD, and dTIMELESS. Recent studies suggest that the clock mechanism in other insect species may differ strikingly from that of Drosophila. We cloned Clock, Bmal, and Timeless homologs (apClock, apBmal, and apTimeless) from the silkmoth Antheraea pernyi, from which a Period homolog (apPeriod) has already been cloned. In Schneider 2 (S2) cell culture assays, apCLOCK:apBMAL activates transcription through an E-box enhancer element found in the 5' region of the apPeriod gene. Furthermore, apPERIOD can robustly inhibit apCLOCK: apBMAL-mediated transactivation, and apTIMELESS can augment this inhibition. Thus, a complete feedback loop, resembling that found in Drosophila, can be constructed from silkmoth CLOCK, BMAL, PERIOD, and TIMELESS. Our results suggest that the circadian autoinhibitory feedback loop discovered in Drosophila is likely to be widespread among insects. However, whereas the transactivation domain in Drosophila lies in the C terminus of dCLOCK, in A. pernyi, it lies in the C terminus of apBMAL, which is highly conserved with the C termini of BMALs in other insects (except Drosophila) and in vertebrates. Our analysis sheds light on the molecular function and evolution of clock genes in the animal kingdom.

ARNTL Transcription Factors↗
PubMed2004

Function of a PscD subunit in a homodimeric reaction center complex of the photosynthetic green sulfur bacterium Chlorobium tepidum studied by insertional gene inactivation. Regulation of energy transfer and ferredoxin-mediated NADP+ reduction on the cytoplasmic side.

The PscD subunit in the homodimeric "type I" photosynthetic reaction center (RC) complex of the green sulfur bacterium Chlorobium tepidum was disrupted by insertional mutagenesis of its relevant pscD gene. This is the first report on the use of the direct mutagenic approach into the RC-related genes in green sulfur bacteria. The RC complex of C. tepidum is supposed to form a homodimer of two identical PscA subunits together with three other subunits: PscB (FA/FB-containing protein), PscC (cytochrome cz), and PscD. PscD shows a relatively low but significant similarity in its amino acid sequence to PsaD in the photosystem I of plants and cyanobacteria. We studied the biochemical and spectroscopic properties of a mutant lacking PscD in order to elucidate its unknown function. 1) The RC complex isolated from the mutant cells showed no band corresponding to PscD on SDS-PAGE analysis. 2) The growth rate of the PscD-less mutant was slower than that of the wild-type cells at low light intensities. 3) Time-resolved fluorescence spectra at 77 K revealed prolonged decay times of the fluorescence from bacteriochlorophyll c on the antenna chlorosome and from bacteriochlorophyll a on the Fenna-Matthews-Olson antenna protein in the mutant cells. The loss of PscD led to a much slower energy transfer from the antenna pigments to the special pair bacteriochlorophyll a (P840). 4) The mutant strain exhibited slightly less activity of ferredoxin-mediated NADP+ photoreduction compared with that in the wild-type strain. The extent of suppression, however, was less significant than that reported in the PsaD-less mutants of cyanobacterial photosystem I. The evolutionary relationship between PscD and PsaD was also discussed based on a structural homology modeling of the former.

Amino Acid Sequence↗
PubMed1996

The unique DNA topology and DNA topoisomerases of hyperthermophilic archaea.

Hyperthermophilic archaea exhibit a unique pattern of DNA topoisomerase activities. They have a peculiar enzyme, reverse gyrase, which introduces positive superturns into DNA at the expense of ATP. This enzyme has been found in all hyperthermophiles tested so far (including Bacteria) but never in mesophiles. Reverse gyrases are formed by the association of a helicase-like domain and a 5'-type 1 DNA topoisomerase. These two domains might be located on the same polypeptide. However, in the methanogenic archaeon Methanopyrus kandleri, the topoisomerase domain is divided between two subunits. Besides reverse gyrase, Archaea contain other type 1 DNA topoisomerases; in particular, M. kandleri harbors the only known procaryotic 3'-type 1 DNA topoisomerase (Topo V). Hyperthermophilic archaea also exhibit specific type II DNA topoisomerases (Topo II), i.e. whereas mesophilic Bacteria have a Topo II that produces negative supercoiling (DNA gyrase), the Topo II from Sulfolobus and Pyrococcus lack gyrase activity and are the smallest enzymes of this type known so far. This peculiar pattern of DNA topoisomerases in hyperthermophilic archaea is paralleled by a unique DNA topology, i.e. whereas DNA isolated from Bacteria and Eucarya is negatively supercoiled, plasmidic DNA from hyperthermophilic archaea are from relaxed to positively supercoiled. The possible evolutionary implications of these findings are discussed in this review. We speculate that gyrase activity in mesophiles and reverse gyrase activity in hyperthermophiles might have originated in the course of procaryote evolution to balance the effect of temperature changes on DNA structure.

Archaea↗
PubMed1995

Construction of murine coronavirus mutants containing interspecies chimeric nucleocapsid proteins.

Targeted RNA recombination was used to construct mouse hepatitis virus (MHV) mutants containing chimeric nucleocapsid (N) protein genes in which segments of the bovine coronavirus N gene were substituted in place of their corresponding MHV sequences. This defined portions of the two N proteins that, despite evolutionary divergence, have remained functionally equivalent. These regions included most of the centrally located RNA-binding domain and two putative spacers that link the three domains of the N protein. By contrast, the amino terminus of N, the acidic carboxy-terminal domain, and a serine- and arginine-rich segment of the central domain could not be transferred from bovine coronavirus to MHV, presumably because these parts of the molecule participate in protein-protein interactions that are specific for each virus (or, possibly, each host). Our results demonstrate that targeted recombination can be used to make extensive substitutions in the coronavirus genome and can generate recombinants that could not otherwise be made between two viruses separated by a species barrier. The implications of these findings for N protein structure and function as well as for coronavirus RNA recombination are discussed.

Amino Acid Sequence↗
PubMed1991

The Saccharomyces cerevisiae TRG1 gene is essential for growth and encodes a lumenal endoplasmic reticulum glycoprotein involved in the maturation of vacuolar carboxypeptidase.

We have isolated and mapped to the left end of chromosome III a single-copy gene (TRG1) encoding a 72-kDa glycoprotein, by screening a yeast genomic library with a DNA probe specifying the catalytic center (APWCGHCK) of thioredoxin-related proteins. the TRG1 gene sequence predicts an amino-terminal leader peptide, two thioredoxin-like domains, five N-glycosylation sites and a carboxyl-terminal HDEL retention signal. The TRG1 protein shows about equal sequence similarity to a mammalian multifunctional protein family residing in the lumen of the endoplasmic reticulum (ER), and to a putative cytosolic alpha form of phosphoinositide-specific phospholipase C. Haploid cells do not survive TRG1 gene disruptions, unless an additional wild-type copy is generated by interchromosomal transposition. Antibodies raised against synthetic amino- and carboxyl-terminal epitopes recognize a pair of lumenl ER glycoproteins (gp70/72) and a cytosolic 48-kDa protein. A 1.8-kilobase TRG1 transcript was translated by a reticulocyte lysate into a 60-kDa protein, which was translocated and processed to a 72-kDa glycoprotein in the presence of ER membrane vesicles. The TRG1 gene was placed under the control of the galactose-inducible and glucose-repressible GAL1 promoter, leading to growth arrest in glucose media. Glucose repression of the TRG1 gene caused the disappearance of gp72 and the accumulation of procarboxypeptidase. Our data indicate that the TRG1 gene encodes a growth essential lumenal ER glycoprotein involved the maturation of vacuolar carboxypeptidase.

Amino Acid Sequence↗
PubMed1985

Structure and exon to protein domain relationships of the mouse carbonic anhydrase II gene.

We have isolated a cosmid clone containing the entire mouse (YBR strain) carbonic anhydrase (CA) II gene in 38 kilobase pairs of genomic DNA. The gene was found to be composed of seven exons and six introns. A TATA box (TATAAAA) and a possible CCAAT box (CCACT) have been located beginning 92 and 142 base pairs, respectively, upstream from the initiation codon ATG. When the regions encoded by exons and protein domains are examined, all but 1 of the 30 putative active site residues are encoded by four exons: exons 2 and 3 mainly code for hydrophilic residues and exons 4 and 6 mostly hydrophobic residues. Two intron splice positions, one between the codons for Glu-116 and Leu-117 and the other interrupting the codon for Gly-143, are located at the bottom of the active site cavity, and the former separates two of the three histidine residues forming ligands to the active site zinc ion. The other four splice sites map to the exterior of the molecule. Thus, except for the possible association of the 29 active site residues encoded by four exons, no obvious correspondence is seen between the regions coded by exons and the functional or secondary structural domains of the mouse CA II molecule. During this study, the possible basis for the two electrophoretic types, CA IIa and CA IIb, of inbred mouse strains was detected as a Gln/His interchange at position 38.

Amino Acid Sequence↗
PubMed1988

Primary structure of T4 DNA polymerase. Evolutionary relatedness to eucaryotic and other procaryotic DNA polymerases.

Bacteriophage T4 gene 43 codes for the viral DNA polymerase. We report here the sequence of gene 43 and about 70 nucleotides of 5'- and 3'-flanking sequences, determined by both DNA and RNA sequencing. We have also purified T4 DNA polymerase from T4 infected Escherichia coli and from E. coli containing a gene 43 overexpression vector. A major portion of the deduced amino acid sequence has been verified by peptide mapping and sequencing of the purified DNA polymerase. All these results are consistent with T4 DNA polymerase having 898 amino acids with a calculated Mr = 103,572. Comparison of the primary structure of T4 DNA polymerase with the sequence of other procaryotic and eucaryotic DNA polymerases indicates that T4 DNA polymerase has regions of striking similarity with animal virus DNA polymerases and human DNA polymerase alpha. Surprisingly, T4 DNA polymerase shares only limited similarity with E. coli polymerase I and no detectable similarity with T7 DNA polymerase. Based on the location of specific mutations in T4 DNA polymerase and the conservation of particular sequences in T4 and eucaryotic DNA polymerases, we propose that the NH2-terminal half of T4 DNA polymerase forms a domain that carries out the 3'----5' exonuclease activity whereas the COOH-terminal half of the polypeptide contains the dNTP-binding site and is necessary for DNA synthesis.

Amino Acid Sequence↗
PubMed2006

The DnaA homolog of the hyperthermophilic eubacterium Thermotoga maritima forms an open complex with a minimal 149-bp origin region in an ATP-dependent manner.

In Escherichia coli, ATP-DnaA, but not ADP-DnaA, forms an initiation complex that undergoes site-specific duplex DNA unwinding, open complex formation. However, it remains unclear how highly the ATP-dependent activation of the initiation factor is conserved in evolution. The hyperthermophile Thermotoga maritima is one of the most ancient eubacteria in evolution. Here, we show that the DnaA homolog (tmaDnaA) of this bacterium forms open complexes with the predicted origin region (tma-oriC) in vitro. TmaDnaA has a strong and specific affinity for ATP/ADP as well as for 12-mer repeating sequences within the tma-oriC. Unlike ADP-tmaDnaA, ATP-tmaDnaA is highly cooperative in DNA binding and forms open complexes in a manner that depends on temperature and the superhelical tension of the tma-oriC-bearing plasmid. The minimal tma-oriC required for unwinding is a 149-bp region containing five repeats of the 12-mer sequence and two AT-rich 9-mer repeats. TmaDnaA-binding to the 12-mer motif provokes DNA bending. The 9-mer region is the duplex-unwinding site. The tmaDnaA-binding and unwinding motifs of tma-oriC share sequence homology with corresponding archaeal and eukaryotic sequences. These findings suggest that the ATP-dependent molecular switch of the initiator and the mechanisms in the replication initiation complex are highly conserved in eubacterial evolution.

Adenosine Triphosphate↗
PubMed1996

Improved green fluorescent protein by molecular evolution using DNA shuffling.

Green fluorescent protein (GFP) has rapidly become a widely used reporter of gene regulation. However, for many organisms, particularly eukaryotes, a stronger whole cell fluorescence signal is desirable. We constructed a synthetic GFP gene with improved codon usage and performed recursive cycles of DNA shuffling followed by screening for the brightest E. coli colonies. A visual screen using UV light, rather than FACS selection, was used to avoid red-shifting the excitation maximum. After 3 cycles of DNA shuffling, a mutant was obtained with a whole cell fluorescence signal that was 45-fold greater than a standard, the commercially available Clontech plasmid pGFP. The expression level in E. coli was unaltered at about 75% of total protein. The emission and excitation maxima were also unchanged. Whereas in E. coli most of the wildtype GFP ends up in inclusion bodies, unable to activate its chromophore, most of the mutant protein is soluble and active. Three amino acid mutations appear to guide the mutant protein into the native folding pathway rather than toward aggregation. Expressed in Chinese Hamster Ovary (CHO) cells, this shuffled GFP mutant showed a 42-fold improvement over wildtype GFP sequence, and is easily detected with UV light in a wide range of assays. The results demonstrate how molecular evolution can solve a complex practical problem without needing to first identify which process is limiting. DNA shuffling can be combined with screening of a moderate number of mutants. We envision that the combination of DNA shuffling and high throughput screening will be a powerful tool for the optimization of many commercially important enzymes for which selections do not exist.

Animals↗
PubMed1990

The dhfrI trimethoprim resistance gene of Tn7 can be found at specific sites in other genetic surroundings.

The dhfrI gene, mediating high-level trimethoprim resistance, was earlier found only on Tn7. Evidence is given here for an alternative location of this gene at a site identical to sites observed earlier for dhfrII on plasmid R388, dhfrV on pLMO20, and aadA on Tn21. All these genes and dhfrI are precisely inserted as discrete GTTA-flanked elements at distinct loci in very conserved surrounding sequences. One of these dhfrI insertions was observed to occur in association with a similarly inserted aadA nucleotidyltransferase gene, which mediates streptomycin and spectinomycin resistance. Close to the insertion site, there is an open reading frame translating into a 337-amino-acid peptide which shows striking similarities to recombinases of the integrase family, sulI, the sulfonamide resistance gene, is very often found close to the insertion point forming a genetic surrounding, originally observed as a part of Tn21-like transposons. The alleged integration mechanism thus provides a recombination pathway for the genetic linkage of sulfonamide and other antibiotic resistance genes, including the most frequently encountered gene for trimethoprim resistance, dhfrI. Furthermore, the newly observed location of dhfrI could shed light on the evolution of the antibiotic resistance region of Tn7, which could be able to take up genes by the same mechanism as that of Tn21-like transposons.

Base Sequence↗
PubMed2002

Comparative genomics of microbial pathogens and symbionts.

We are interested in quantifying the contribution of gene acquisition, loss, expansion and rearrangements to the evolution of microbial genomes. Here, we discuss factors influencing microbial genome divergence based on pair-wise genome comparisons of closely related strains and species with different lifestyles. A particular focus is on intracellular pathogens and symbionts of the genera Rickettsia, Bartonella and BUCHNERA: Extensive gene loss and restricted access to phage and plasmid pools may provide an explanation for why single host pathogens are normally less successful than multihost pathogens. We note that species-specific genes tend to be shorter than orthologous genes, suggesting that a fraction of these may represent fossil-orfs, as also supported by multiple sequence alignments among species. The results of our genome comparisons are placed in the context of phylogenomic analyses of alpha and gamma proteobacteria. We highlight artefacts caused by different rates and patterns of mutations, suggesting that atypical phylogenetic placements can not a priori be taken as evidence for horizontal gene transfer events. The flexibility in genome structure among free-living microbes contrasts with the extreme stability observed for the small genomes of aphid endosymbionts, in which no rearrangements or inflow of genetic material have occurred during the past 50 millions years (1). Taken together, the results suggest that genomic stability correlate with the content of repeated sequences and mobile genetic elements, and thereby indirectly with bacterial lifestyles.

Alphaproteobacteria↗
PubMed1981

The density distribution of gene loci over the genetic map of Escherichia coli: its structural, functional and evolutionary implications.

A quantitative analysis was carried out on the dispersion of gene loci over the E. coli genetic map. Therefore, the map was divided into regions characterized by an homogeneous gene density. This created a distribution pattern of gene loci that contained a symmetry axis located near to the origin of DNA replication. The pattern could be subdivided into a set of 22 functional domains containing gene loci whose products revealed a biochemical or functional relatedness. A correlation was found between the boundary positions of these domains and the distribution of F plasmid- and DNA insertion sites over the E. coli chromosome. The structural, functional and evolutionary implications of these findings are discussed.

Biological Evolution↗