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A traC mutant that retains sensitivity to f1 bacteriophage but lacks F pili.

An F lac pro mutant which was temperature sensitive for infection by the filamentous bacteriophage f1 but resistant to the F-specific icosahedral RNA phage f2 was isolated. Cells carrying the F' mutation failed to elaborate F pili at all temperatures. Mutant cells were able to pair with recipient cells during bacterial conjugation, but transfer of conjugal DNA occurred at a greatly reduced frequency. Complementation analyses showed the F' mutation to be in the traC gene. When a plasmid carrying traC was introduced into hosts harboring the F' mutation, phage sensitivity, the ability to elaborate F pili, and conjugation efficiency were restored. The mutation was named traC1044. The F lac pro traC1044 mutant appears to be unique among traC mutants in retaining host sensitivity to the filamentous phage f1 in the absence of expression of extended F pili. Phage f1 attachment sites appeared to be present at the cell surface in traC1044 mutants. The reduced accessibility of these sites may account for the reduced efficiency of phage f1 infection of traC1044 hosts, although the possibility that a defect was present in the receptor site itself was not eliminated. Membranes of hosts carrying the F' mutation contained a full complement of mature F-pilin subunits, so the product of traC is presumably required for pilus assembly but not for pilin processing. This, together with the deficiency in conjugal DNA transfer, suggests that traC may be part of a membrane-spanning tra protein complex responsible for pilus assembly and disassembly and conjugal DNA transmission.

Chromosome Mapping↗

Complete mitochondrial DNA sequence of Conger myriaster (Teleostei: Anguilliformes): novel gene order for vertebrate mitochondrial genomes and the phylogenetic implications for anguilliform families.

The complete nucleotide sequence of the mitochondrial genome was determined for a conger eel, Conger myriaster (Elopomorpha: Anguilliformes), using a PCR-based approach that employs a long PCR technique and many fish-versatile primers. Although the genome [18,705 base pairs (bp)] contained the same set of 37 mitochondrial genes [two ribosomal RNA (rRNA), 22 transfer RNA (tRNA), and 13 protein-coding genes] as found in other vertebrates, the gene order differed from that recorded for any other vertebrates. In typical vertebrates, the ND6, tRNA(Glu), and tRNA(Pro) genes are located between the ND5 gene and the control region, whereas the former three genes, in C. myriaster, have been translocated to a position between the control region and the tRNA(Phe) gene that are contiguously located at the 5' end of the 12S rRNA gene in typical vertebrates. This gene order is similar to the recently reported gene order in four lineages of birds in that the latter lack the ND6, tRNA(Glu), and tRNA(Pro) genes between the ND5 gene and the control region; however, the relative position of the tRNA(Pro) to the ND6-tRNA(Glu) genes in C. myriaster was different from that in the four birds, which presumably resulted from different patterns of tandem duplication of gene regions followed by gene deletions in two distantly related groups of organisms. Sequencing of the ND5-cyt b region in 11 other anguilliform species, representing 11 families, plus one outgroup species, revealed that the same gene order as C. myriaster was shared by another 4 families, belonging to the suborder Congroidei. Although the novel gene orders of four lineages of birds were indicated to have multiple independent origins, phylogenetic analyses using nucleotide sequences from the mitochondrial 12S rRNA and cyt b genes suggested that the novel gene orders of the five anguilliform families had originated in a single ancestral species.

Amino Acid Sequence↗

[Processing of tRNA].

The article reviews various aspects of tRNA biosynthesis in pro- and eukaryotes. Some data on the tRNA gene localization are presented. Structures of tRNA precursors are given; their conversion into mature tRNA molecules and the corresponding enzymatic systems are discussed in detail. Special emphasis is given to the transcription of tRNA genes and processing of tRNA precursors in vitro. Similarities and diversities of tRNA processing in pro- and eukaryotes are discussed.

Animals↗

Mechanism of RNase T1: concerted triester-like phosphoryl transfer via a catalytic three-centered hydrogen bond.

BACKGROUND: The microscopic events of ribonuclease (RNase) catalyzed phosphoryl transfer reactions are still a matter of debate in which the contenders adhere to either the classical concerted acid-base mechanism or a more sequential triester-like mechanism. In the case of RNase A, small thio-effects of the nonbridging oxygens have been invoked in favor of the classical mechanism. However, the RNase T1 catalyzed transphosphorylation of phosphorothioate RNA is highly stereoselective. R(P) thio-substituted RNA is depolymerized 60000 times faster than S(P) thio-substituted RNA by this enzyme, whereas the uncatalyzed cleavage of both substrates occurs at comparable rates. We combined site-directed mutagenesis in the RNase active site and stereospecific thio-substitution of an RNA substrate to probe the intermolecular interactions of the enzyme with the nonbridging pro-S(P) oxygen that bring about this stereoselectivity of RNase T1. RESULTS: Thio-substitution of the nonbridging pro-S(P) oxygen in the substrate afflicts chemical turnover but not ground state binding whereas thio-substitution of the nonbridging pro-R(P) oxygen does not affect the kinetics of RNase T1. Site-directed mutagenesis of the catalytic base Glu58 impairs the enzyme's ability to discriminate both phosphorothioate diastereomers. Glu58Ala RNase T1 cleaves R(P) and S(P) phosphorothioate RNA with similar rates. The dependence of the pro-S(P) thio-effect on the presence of the Glu58 carboxylate evidences a strong rate-limiting interaction between the nonbridging pro-S(P) oxygen and the catalytic base Glu58 in the wild type enzyme. CONCLUSIONS: Based on these results, we put forward a new triester-like mechanism for the RNase T1 catalyzed reaction that involves a three-centered hydrogen bond between the 2'-OH group, the nonbridging pro-S(P) oxygen and one of the carboxylate oxygens of Glu58. This interaction allows nucleophilic attack on an activated phosphate to occur simultaneously with general base catalysis, ensuring concerted phosphoryl transfer via a triester-like mechanism.

Binding Sites↗

Ectopic pro-opiolipomelanocortin: sequence of cDNA coding for beta-melanocyte-stimulating hormone and beta-endorphin.

A recombinant bacterial plasmid, pMS1, was constructed that contains 318 nucleotides complementary to a portion of pro-opiolipomelanocortin (proOLMC) messenger RNA from an ectopic adrenocorticotropin-producing tumor. The cloned complementary DNA insert, which contains the sequence that codes for all of the beta-melanocyte-stimulating hormone and beta-endorphin portions of proOLMC, as well as the 3' nontranslated section, is identical to the genomic sequence. Hybridization of tumor proOLMC complementary DNA to RNA subjected to electrophoresis and transferred to a nitrocellulose filter revealed two proOLMC messenger RNA species in the tumor polyadenylated RNA, but only one in pituitary polyadenylated RNA. At least one of the tumor proOLMC messenger RNA's is similar, if not identical, to human pituitary proOLMC messenger RNA.

Amino Acid Sequence↗

Evolutionary implications of phylogenetic analyses of the gene transfer agent (GTA) of Rhodobacter capsulatus.

The gene transfer agent (GTA) of the a-proteobacterium Rhodobacter capsulatus is a cell-controlled genetic exchange vector. Genes that encode the GTA structure are clustered in a 15-kb region of the R. capsulatus chromosome, and some of these genes show sequence similarity to known bacteriophage head and tail genes. However, the production of GTA is controlled at the level of transcription by a cellular two-component signal transduction system. This paper describes homologues of both the GTA structural gene cluster and the GTA regulatory genes in the a-proteobacteria Rhodopseudomonas palustris, Rhodobacter sphaeroides, Caulobacter crescentus, Agrobacterium tumefaciens and Brucella melitensis. These sequences were used in a phylogenetic tree approach to examine the evolutionary relationships of selected GTA proteins to these homologues and (pro)phage proteins, which was compared to a 16S rRNA tree. The data indicate that a GTA-like element was present in a single progenitor of the extant species that contain both GTA structural cluster and regulatory gene homologues. The evolutionary relationships of GTA structural proteins to (pro)phage proteins indicated by the phylogenetic tree patterns suggest a predominantly vertical descent of GTA-like sequences in the a-proteobacteria and little past gene exchange with (pro)phages.

Alphaproteobacteria↗

Nucleic acid transfer through cell membranes: towards the underlying mechanisms.

Various cases of DNA (RNA) transfer through membranes of living cells are reviewed. They are classified into two major categories: those which occur in Nature (natural transfer) and those imposed by various physical and chemical treatments of cells (induced transfer). Among the examples of natural transfer surveyed are the transfer during bacterial conjugation, genetic transformation, viral infection of bacteria, and nuclear membrane trafficking. Consideration of the induced transfer is focused on the two methods most widely used at present to introduce foreign genetic information into pro- and eukaryotic cells: Ca2+ (and some other divalent cations)-induced and calcium phosphate-induced transfer, and transfer during electroporation of cells. Emphasis is made on the underlying mechanisms of transfer, or rather on what is currently known about them. Energetic aspects of transfer are also discussed and different tentative models of transfer are presented.

Bacteria↗

Aneurysm or occlusive disease--factors determining the clinical course of atherosclerosis of the infrarenal aorta.

Atherosclerosis of the infrarenal aorta results in distinct clinical entities--aortoiliac occlusive disease (AOD) and abdominal aortic aneurysm (AAA). Although loss of collagen has been implicated in AAA, collagen accumulation plays a role in AOD. In vivo collagen-gene expression can be assessed using complementary DNA for collagen types I and III alpha-chains. The purpose of this study is to compare total collagen (type I + III) and collagen types I and III messenger RNA in AAA, AOD and normal aorta. Specimens were collected from the infrarenal aorta during operation for AOD (n = 7), AAA (n = 7), autopsy, or organ procurement (normal; n = 7). Northern transfer analysis of total RNA was used to compare mRNA levels for type I and III collagen. After preliminary extraction, specimens were hydrolyzed for hydroxyproline analysis used to calculate total collagen (type I + III). Relative levels of type I (pro-a1[1]) mRNA were greater in both AOD (0.77 +/- 0.35) and AAA tissue (0.94 +/- 0.24; p = 0.6) than in normal aorta (0.02 +/- 0.03). Type III (pro-a1[III]) mRNA levels were also greater in AOD (2.52 +/- 0.19; p = 0.09) and AAA tissue (3.15 +/- 1.3) than in normals (0.97 +/- 0.47). Total collagen concentration was increased in AOD (45.6% +/- 3.1% dry weight; p less than 0.05) but not AAA tissue (27.8% +/- 4%) when compared to normal aorta (34.7% +/- 2.3%). Collagen type I and III gene expression is greater in older, diseased aorta, yet collagen accumulated only in AOD. This implies a similar synthetic response in both AOD and AAA. Thus, proteolytic degradation in AAA appears to determine collagen content and possibly the clinical course of the atherosclerotic process.

Aged↗

Structure and organization of a cluster of sic tRNA genes in the space between tandem ribosomal RNA gene sets in Bacillus subtilis.

Hybridization of Southern blots of EcoRI digests of total Bacillus subtilis DNA with ribosomal RNA and transfer RNA probes provides evidence of highly clustered tRNA and rRNA genes, with several tRNA clusters being located in spaces which span between tandem ribosomal RNA gene sets. Clones containing these tRNA clusters were isolated from a Charon 4A library. One of them, denoted trrnB, was partially sequenced. A cluster of six tRNA genes was found, with anticodon assignments of Asn, Thr, Gly, Arg, Pro, Ala. This cluster is closely flanked on both sides by ribosomal RNA gene sets, which were identified by sequencing upward through the 5 S rRNA gene, across a space, and into the 23 S rRNA gene, and also sequencing downward into the 16 S rRNA gene. The tRNA gene cluster appears to be organized into at least two transcriptional units separated by an attenuator region. These transcriptional units may be components of the flanking ribosomal RNA operons. The putative promoter region of the downstream 16 S rRNA is organized differently from Escherichia coli; it is smaller and seems less complex. This gene organization provides insight into possible mechanisms for coordinate and differential control of transfer and ribosomal RNA gene expression.

Anticodon↗

Transfer of primer binding site-mutated simian immunodeficiency virus vectors by genetically engineered artificial and hybrid tRNA-like primers.

Simian immunodeficiency viruses (SIV) harbor primer binding sites (PBS) matching tRNA or tRNA. To study determinants of primer usage in SIV, a SIVmac239-based vector was impaired by mutating the PBS to a sequence (PBS-X2) with no match to any tRNA. By cotransfection of a synthetic gene encoding a tRNA(Pro)-like RNA with a match to PBS-X2, the activity of this vector could be restored to a transduction efficiency slightly lower than that of the wild-type vector. A vector with a PBS matching tRNA(Pro) was functional at a level slightly below that of the wild-type vector, but higher transduction efficiency could be obtained by cotransfection of a gene for an engineered tRNA(Pro)-tRNA hybrid with a match to PBS-Pro. The importance of tRNA backbone identity was further analyzed by complementing the PBS-X2 vector with a gene for a matching x2 primer with a tRNA backbone, which led to three- to fourfold-higher titers than those observed for the x2 primer with the tRNA(Pro) backbone. In summary, our results demonstrate flexibility in PBS and primer usage for SIVmac239, with PBS-primer complementarity being the major determinant, in analogy with previous findings for murine leukemia viruses and human immunodeficiency virus type 1.

Animals↗

Effect of glucose starvation on the expression of transferred tsx genes in Escherichia coli K12 zygotes.

Escherichia coli K12 Hfr H Tsxs Strs and F- Pro- Tsxr His- Arg- Strr bacteria were conjugated in the absence of arginine with or without glucose. The efficiency of conjugation, measured by the frequency of Pro+ and His+ recombinants was not affected. Arginine starvation alone did not affect the tsxs gene expression which occurred in all the zygotes which had received the gene. In contrast, argine and glucose starvation allows tsxs expression only in those zygotes in which the donor gene had been integrated in the genome. As the glucose starvation brings on a destabilization of the messenger RNA synthesized by the F- cells in absence of arginine, the results can be interpreted as follows: the transferred tsxs genes are transitorily expressed in all the zygotes at the unintegrated state. After this transient period, only thsoe genes integrated in the chromosomes of the zygotes continue to be expressed.

Arginine↗

Organization of the Mitochondrial Genome of a Deep-Sea Fish, Gonostoma gracile (Teleostei: Stomiiformes): First Example of Transfer RNA Gene Rearrangements in Bony Fishes.

: We determined the complete nucleotide sequence of the mitochondrial genome (except for a portion of the putative control region) for a deep-sea fish, Gonostoma gracile. The entire mitochondrial genome was purified by gene amplification using long polymerase chain reaction (long PCR), and the products were subsequently used as templates for PCR with 30 sets of newly designed, fish-universal primers that amplify contiguous, overlapping segments of the entire genome. Direct sequencing of the PCR products showed that the genome contained the same 37 mitochondrial structural genes as found in other vertebrates (two ribosomal RNA, 22 transfer RNA, and 13 protein-coding genes), with the order of all rRNA and protein-coding genes, and 19 tRNA genes being identical to that in typical vertebrates. The gene order of the three tRNAs (tRNA(Glu), tRNA(Thr), and tRNA(Pro)) relative to cytochrome b, however, differed from that determined in other vertebrates. Two steps of tandem duplication of gene regions, each followed by deletions of genes, can be invoked as mechanisms generating such rearrangements of tRNAs. This is the first example of tRNA gene rearrangements in a bony fish mitochondrial genome.

Journal Article↗

Sequence fossils, triplet expansion, and reconstruction of earliest codons.

mRNA sequences are known to carry a hidden periodical pattern (GCU)n, which may be considered a remnant of sequence organization of mRNA early in its evolution, dominated by codons for alanine and their point mutation derivatives. A similar pattern is characteristic of the master (consensus) tRNA sequence derived in 1981 by Eigen and Winkler-Oswatitsch. The master tRNA sequence is thought to represent one of the earliest mRNA. From analysis of literature and from our own calculations presented in this work, the (GCU)n pattern appears to be the most expandable in the norm and in disease. The speculation is put forward that (GCU)n and polyalanine have been key players at the beginning of the triplet code, and the first codons, apart from the GCU triplet, were point change derivatives of the generic triplet GCU, coding for amino acids present in the early prebiotic-biotic environment. The set of the earliest amino acids is derived on the basis of structural simplicity, presence in imitated prebiotic conditions and involvement with class II aminoacyl-tRNA synthetases. The set consists of six amino acids: Ala, Asp, Gly, Pro, Ser and Thr. All these amino acids are, indeed, encoded by the GCU triplet and its derivatives, as predicted. Thus, the pairs GCN (Ala), GAU (Asp), GGU (Gly), CCU (Pro), UCU (Ser) and ACU (Thr) can be viewed as an early triplet code.

Codon↗

One polypeptide with two aminoacyl-tRNA synthetase activities.

The genome sequences of certain archaea do not contain recognizable cysteinyl-transfer RNA (tRNA) synthetases, which are essential for messenger RNA-encoded protein synthesis. However, a single cysteinyl-tRNA synthetase activity was detected and purified from one such organism, Methanococcus jannaschii. The amino-terminal sequence of this protein corresponded to the predicted sequence of prolyl-tRNA synthetase. Biochemical and genetic analyses indicated that this archaeal form of prolyl-tRNA synthetase can synthesize both cysteinyl-tRNA(Cys) and prolyl-tRNA(Pro). The ability of one enzyme to provide two aminoacyl-tRNAs for protein synthesis raises questions about concepts of substrate specificity in protein synthesis and may provide insights into the evolutionary origins of this process.

Amino Acyl-tRNA Synthetases↗

Molecular recognition of threonine tRNA by threonyl-tRNA synthetase from an extreme thermophilic archaeon, Aeropyrum pernix K1.

To investigate the recognition sites of tRNA(Thr) for threonyl-tRNA synthetase (ThrRS) from an extreme thermophilic and aerobic archaeon, Aeropyrum pernix K1, threonylation experiments using various in vitro mutant transcripts of tRNA(Thr) were examined. The results indicated that A. pernix ThrRS did recognize the first three base pairs of acceptor stem in addition to the second and the third letters of anticodon of tRNA(Thr), in spite of its N-terminal truncated unique structure. Discriminator base was not involved in recognition by A. pernix ThRS. These determinants were confirmed by the identity switching experiments from the in vitro mutants of A. pernix tRNA(Pro) and tRNA(Asn).

Archaea↗

The use of a double subgenomic Sindbis virus expression system to study mosquito gene function: effects of antisense nucleotide number and duration of viral infection on gene silencing efficiency.

Recently we established a simple, effective antisense strategy using a double subgenomic Sindbis (dsSIN) virus expression system to study gene function in mosquitoes. In this study, we further elucidate the effects of antisense nucleotide number and duration of viral infection on mosquito gene silencing efficiency by the dsSIN virus expression system. Over 15 days post virus infection, the degree of parasite melanization was progressively reduced by more than 95%, 75% and 55% in the mosquito Armigeres subalbatus transduced with 600, 147 or 36 bases antisense RNA, targeted to the highly conserved copper binding region of the Ar. subalbatus prophenoloxidase I gene (As-pro-POI), respectively. As the duration of viral infection increased from day 3-15, the degree of parasite melanization progressively decreased in all mosquitoes transduced with antisense RNA, irrespective of the lengths of antisense RNA. Progressive loss of parasite melanization function was found to correlate with down regulation of As-pro-PO expression at both the mRNA and protein activity levels, and reductions in virus titres in mosquitoes transduced with antisense RNA. A small pro-PO RNA (c. twenty-five nucleotides) was identified in mosquitoes transduced with antisense RNA. These data suggest that As-pro-POI gene expression is knocked down by degrading the As-pro-POI mRNA through the RNAi pathway. In conclusion, our study demonstrates that even a short antisense RNA (thirty-six bases) can cause silencing of the As-pro-POI gene, and the effects of endogenous gene silencing by dsSIN expression system on mosquito gene functions can be accumulative.

Animals↗

Plasma cholesteryl ester transfer protein and high-density lipoproteins: new insights from molecular genetic studies.

Recent studies in transgenic mice provide strong evidence for a direct anti-atherogenic role of high-density lipoproteins (HDL) and highlight the importance of multiple gene interactions in the regulation of HDL levels. Plasma lipid transfer processes mediated by cholesteryl ester transfer protein (CETP) have a major impact on HDL levels, as revealed in studies of human genetic CETP deficiency and CETP transgenic mice. Subsequent to the discovery of an intron 14 CETP gene splicing defect, several new CETP gene mutations have been discovered recently in Japanese and other populations. One of these is an exon 15 missense mutation, changing amino acid 442 of CETP from aspartate to glycine. Population studies in Japan indicate that CETP gene mutations are sufficiently common to have a significant influence on HDL levels in the general population. Studies in transgenic mice show that CETP expression results in decreased levels of HDL cholesterol, but that the effects of CETP on HDL apolipoprotein A-I (apoA-I) content and size show important modulation by co-expression with transgenes encoding human apoA-I, apoC-III and apoA-II. In addition to the apparent antiatherogenic phenotype of human genetic CETP deficiency, high level expression of CETP in transgenic mice leads to accelerated atherosclerosis, illustrating the pro-atherogenic potential of CETP expression.

Animals↗

Sequence analysis of 12 structural genes and a novel non-coding region from mitochondrial DNA of Atlantic cod, Gadus morhua.

We have determined the nucleotide sequences of 12 structural genes from the mitochondrial DNA of Atlantic cod, Gadus morhua. These genes encode the proteins NADH dehydrogenase subunit 2, cytochrome c oxidase subunit I, cytochrome c oxidase subunit II, and apocytochrome b, as well as the transfer RNAs tRNA(Ile), tRNA(Gln), tRNA(Met), tRNA(Ser) (UCN), tRNA(Asp), tRNA(Glu), tRNA(Thr) and tRNA(Pro). The apocytochrome b sequences were used to construct a phylogenetic tree revealing the evolutionary divergence between modern bony fishes, sturgeon and sharks. We found that bony fishes display the same slow amino acid substitution rates in the mitochondrial encoded proteins as cartilaginous fishes (sharks). A novel non-coding region of 74 base pairs not found in other fishes where sequence data are available is located between the genes encoding tRNA(Thr) and tRNA(Pro). This region contains both direct and inverted repeat motifs that may function in termination of the H-strand transcript.

Amino Acid Sequence↗