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PubMed1992

Molecular analysis of the Haemophilus ducreyi groE heat shock operon.

Chancroid is a sexually transmitted genital ulcer disease caused by Haemophilus ducreyi. Previously, we developed diagnostic DNA probes for H. ducreyi (L. M. Parsons, M. Shayegani, A. L. Waring, and L. H. Bopp, J. Clin. Microbiol. 27:1441-1445, 1989). In the present study, DNA sequencing of one of the diagnostic probes revealed two adjacent open reading frames (ORFs). These H. ducreyi ORFs and the encoded proteins show significant homology with the groE genes and GroES and GroEL heat shock proteins from several bacterial pathogens and with conserved eukaryotic 60-kDa heat shock proteins. The first H. ducreyi ORF (groES) is preceded by sequences similar to those of the Escherichia coli consensus heat shock promoters and is 288 nucleotides long and is capable of encoding a protein of 10.3 kDa. The second ORF (groEL) is 1,641 nucleotides long and is capable of encoding a protein of 57.8 kDa. Northern (RNA blot) analysis demonstrated the presence of a high level of groE mRNA in exponential-phase H. ducreyi grown in hemin broth at the organism's optimal growth temperature (33 degrees C), with increased levels seen following heat shock. Heat shock also increased the thermostability of the organisms, since stressed cells were more resistant to the lethal effects of rapid chilling. Electrophoretic analysis and immunoblots demonstrated that the predominant protein produced by exponential-phase H. ducreyi was a heat-inducible, immunoreactive protein of approximately 60 kDa (GroEL). Also, H. ducreyi groE mRNA and GroEL were expressed and inducible by heat in E. coli. This is the first report describing the cloning, sequencing, and expression of H. ducreyi protein-encoding genes.

Amino Acid Sequence↗
PubMed2004

Mutation scanning of the androgen receptor gene in patients with psychiatric disorders reveals highly conserved variants in alcoholic and phobia patients.

Sex steroids exert potent effects on mood and mental state in humans. They may contribute to the risk of psychiatric disorders. To investigate this hypothesis, coding and splice junction sequences of the androgen receptor gene were scanned in genomic DNA samples to search for variants affecting protein structure and expression (VAPSEs). Ninety-six schizophrenics, along with pilot samples of patients with bipolar disorder, attention-deficit hyperactivity disorder, alcoholism and autism were analyzed with DOVAM-S, a robotically enhanced, optimized form of single-strand conformation polymorphism analysis. A total of 669 kb of genomic sequence was analyzed. Two VAPSEs were identified: R726L was found in one of 17 scanned alcoholics, and P516S, a novel VAPSE, was identified in one of three phobia patients. There were no length trends of the CAG triplets associated with schizophrenia. R726L and P516S occur at highly conserved amino acids. Further study is required to assess whether these VAPSEs contribute to the risk of alcoholism or phobia or other diseases.

Alcoholism↗
PubMed2001

Automated constant denaturant capillary electrophoresis applied for detection of KRAS exon 1 mutations.

In this study, we have applied automated constant denaturant capillary electrophoresis (ACDCE) for the detection of KRAS exon 1 mutations. Samples from 191 sporadic colon carcinomas previously analyzed for KRAS mutations with allele-specific PCR (ASPCR), temporal temperature gradient electrophoresis (TTGE), and constant denaturant capillary electrophoresis (CDCE) were analyzed. In ACDCE, an unmodified ABI Prism 310 genetic analyzer with constant denaturant conditions separated fluorescein-labeled PCR products. Temperature in combination with a chemical denaturant was used for separation. The optimal separation conditions for PCR-amplified KRAS exon 1 fragments were determined by adjusting the temperature before electrophoresis. In the ACDCE analysis, the sequence of a mutant was determined by comparing the electropherogram of the fragment to that of known mutations followed by mixing the sample with control mutations before reanalysis. In a titration experiment mixing mutant and wild-type alleles, the sensitivity for mutation detection was shown to be 0.6% in this automated CDCE technique. The automation of CDCE allowed rapid analysis of a large number of test samples over as short period of time and with a commercially available apparatus.

Autoanalysis↗
PubMed2001

Comparative mutational analysis of cis-acting RNA signals for translational frameshifting in HIV-1 and HTLV-2.

Human immunodeficiency virus type 1 (HIV-1) and human T cell leukemia virus type II (HTLV-2) use a similar mechanism for -1 translational frameshifting to overcome the termination codon in viral RNA at the end of the gag gene. Previous studies have identified two important RNA signals for frameshifting, the slippery sequence and a downstream stem-loop structure. However, there have been somewhat conflicting reports concerning the individual contributions of these sequences. In this study we have performed a comprehensive mutational analysis of the cis-acting RNA sequences involved in HIV-1 gag-pol and HTLV-2 gag-pro frameshifting. Using an in vitro translation system we determined frameshifting efficiencies for shuffled HIV-1/HTLV-2 RNA elements in a background of HIV-1 or HTLV-2 sequences. We show that the ability of the slippery sequence and stem-loop to promote ribosomal frameshifting is influenced by the flanking upstream sequence and the nucleotides in the spacer element. A wide range of frameshift efficiency rates was observed for both viruses when shuffling single sequence elements. The results for HIV-1/HTLV-2 chimeric constructs represent strong evidence supporting the notion that the viral wild-type sequences are not designed for maximal frameshifting activity but are optimized to a level suited to efficient viral replication.

Base Sequence↗
PubMed2000

Gene structure prediction by spliced alignment of genomic DNA with protein sequences: increased accuracy by differential splice site scoring.

Gene identification in genomic DNA from eukaryotes is complicated by the vast combinatorial possibilities of potential exon assemblies. If the gene encodes a protein that is closely related to known proteins, gene identification is aided by matching similarity of potential translation products to those target proteins. The genomic DNA and protein sequences can be aligned directly by scoring the implied residues of in-frame nucleotide triplets against the protein residues in conventional ways, while allowing for long gaps in the alignment corresponding to introns in the genomic DNA. We describe a novel method for such spliced alignment. The method derives an optimal alignment based on scoring for both sequence similarity of the predicted gene product to the protein sequence and intrinsic splice site strength of the predicted introns. Application of the method to a representative set of 50 known genes from Arabidopsis thaliana showed significant improvement in prediction accuracy compared to previous spliced alignment methods. The method is also more accurate than ab initio gene prediction methods, provided sufficiently close target proteins are available. In view of the fast growth of public sequence repositories, we argue that close targets will be available for the majority of novel genes, making spliced alignment an excellent practical tool for high-throughput automated genome annotation.

Algorithms↗
PubMed1996

An alkaline D-stereospecific endopeptidase with beta-lactamase activity from Bacillus cereus.

We purified a novel extracellular D-stereospecific endopeptidase, alkaline D-peptidase (D-stereospecific peptide hydrolase, EC 3.4.11.-), to homogeneity from the culture broth of the soil bacterium Bacillus cereus strain DF4-B. The Mr of the enzyme was 37,952, and it was composed of a single polypeptide chain. The optimal pH for activity was approximately 10.3. The enzyme was strictly D-stereospecific toward oligopeptides composed of Dphenylalanine such as (D-Phe)3 and (D-Phe)4. The enzyme also acted to a lesser extent on (D-Phe)6, Boc-(D-Phe)4 (where Boc is tert-butoxycarbonyl), Boc-(D-Phe)4 methyl ester, Boc-(D-Phe)3 methyl ester, Boc-(D-Phe)2, (D-Phe)2, and others, but not upon their corresponding peptides composed of L-Phe, (D-Ala)n (n = 2-5), (D-Val)3, and (D-Leu)2. The mode of action of the enzyme was clarified with synthetic substrates ((D-Phe)2-D-Tyr and D-Tyr-(D-Phe)2) and eight stereoisomers of (Phe)3. The enzyme had beta-lactamase activity toward ampicillin and penicillin G, although carboxypeptidase DD and D-aminopeptidase activities were undetectable. The gene coding for alkaline D-peptidase (adp) was cloned into plasmid pUC118, and a 1164-base pair open reading frame consisting of 388 codons was identified as the adp gene. The predicted polypeptide was similar to carboxypeptidase DD from Streptomyces R61, penicillin-binding proteins from Streptomyces lactamdurans and Bacillus subtilis, and class C beta-lactamases. Thus, the enzyme was categorized as a new "penicillin-recognizing enzyme."

Amino Acid Sequence↗
PubMed2006

Selection and persistence of non-nucleoside reverse transcriptase inhibitor-resistant HIV-1 in patients starting and stopping non-nucleoside therapy.

BACKGROUND: Understanding the selection and decay of drug-resistant HIV-1 variants is important for designing optimal antiretroviral therapy. OBJECTIVE: To develop a high-throughput, real-time reverse transcriptase (RT) polymerase chain reaction (PCR) assay to quantify non-nucleoside reverse transcriptase inhibitor (NNRTI)-resistant variants K103N (AAT or AAC alleles) at frequencies as low as 0.1%, and to apply this to monitor these variants before, during, and after NNRTI therapy. METHODS: HIV-1 RNA in longitudinal plasma samples obtained from patients starting and stopping NNRTI therapy was converted to cDNA and the target sequence region amplified and quantified by real-time PCR. Approximately 10 copies/reaction provided a template for a second round of PCR using primers that discriminated between the mutant and wild-type alleles. Amplification specificity was confirmed by thermal denaturation analysis. RESULTS: Frequencies of 103N similar to assay background (0.029%) were observed in longitudinal samples from 9 of 12 treatment-naive patients; three patients had transient increases in 103N frequency to a range of 0.21-0.48%, which was 7-16.5 times assay background. Analysis of longitudinal plasma samples from six NNRTI-experienced patients showed three patterns: persistence of 103N variants after stopping NNRTI therapy, codon switching of 103N between AAC and AAT during NNRTI therapy, and decay of 103N variants to below assay background after cessation of NNRTI therapy. CONCLUSIONS: Allele-specific RT-PCR quantified the emergence and decay of drug-resistant variants in patients over a broad range of frequencies (0.1-100%). The rate of decay of K103N variants after stopping NNRTI therapy was highly variable.

Adult↗
PubMed1992

Memory T cells of a patient with follicular thyroid carcinoma recognize peptides derived from mutated p21 ras (Gln-->Leu61).

Point mutations in ras genes resulting in substitutions of amino acid Gly in positions 12 and 13, and Gln in position 61 of the ras gene product p21, are commonly found in human tumors. Peptides derived from aberrant p21 may elicit a tumor specific T cell response, provided that these peptides can bind to HLA molecules of the tumor and the patient has T cells able to recognize the corresponding peptide-HLA complex. Here we report that CD4+ T cells of memory type (CD45RO+) from a patient with a follicular thyroid carcinoma respond against a synthetic peptide derived from aberrant p21 ras having a Gln-->Leu substitution at position 61. Such responses were not observed when T cells from healthy volunteers or cancer patients where this mutation does not usually occur were stimulated with this peptide. The responding T cells did not cross-react with the corresponding peptide derived from native p21 ras nor did they recognize peptides carrying other substitutions in position 61. T cells clones were generated which recognized this Leu61 peptide when presented by HLA-DQ8 molecules. These T cell clones also recognized the corresponding intact p21 ras protein. By using several different synthetic peptides, a peptide with optimal stimulatory capacity was defined. Performing polymerase chain reaction and oligonucleotide probing we were, however, not able to detect the p21 ras gene encoding the Gln-->Leu substitution in DNA from tumor biopsies from the patient. This may indicate that tumor cells harboring the mutation leading to the Gln-->Leu substitution had been eliminated and that tumor progression was due to cells that had deleted the mutated ras gene. The finding that ras derived peptides and recombinant mutated p21 ras are immunogenic in man may form the basis for the development of cancer immunotherapy based on synthetic oncogene derived peptides.

Adenocarcinoma↗
PubMed1996

Interaction of Sp1 with the growth- and cell cycle-regulated transcription factor E2F.

Within the region around 150 bp upstream of the initiation codon, which was previously shown to suffice for growth-regulated expression, the murine thymidine kinase gene carries a single binding site for transcription factor Sp1; about 10 bp downstream of this site, there is a binding motif for transcription factor E2F. The latter protein appears to be responsible for growth regulation of the promoter. Mutational inactivation of either the Sp1 or the E2F site almost completely abolishes promoter activity, suggesting that the two transcription factors interact directly in delivering an activation signal to the basic transcription machinery. This was verified by demonstrating with the use of glutathione S-transferase fusion proteins that E2F and Sp1 bind to each other in vitro. For this interaction, the C-terminal part of Sp1 and the N terminus of E2F1, a domain also present in E2F2 and E2F3 but absent in E2F4 and E2F5, were essential. Accordingly, E2F1 to E2F3 but not E2F4 and E2F5 were found to bind sp1 in vitro. Coimmunoprecipitation experiments showed that complexes exist in vivo, and it was estabilished that the distance between the binding sites for the two transcription factors was critical for optimal promoter activity. Finally, in vivo footprinting experiments indicated that both the sp1 and E2F binding sites are occupied throughout the cell cycle. Mutation of either binding motif abolished binding of both transcription factors in vivo, which may indicate cooperative binding of the two proteins to chromatin-organized DNA. Our data are in line with the hypothesis that E2F functions as a growth- and cell cycle regulated tethering factor between Sp1 and the basic transcription machinery.

3T3 Cells↗
PubMed1999

Physicochemical optimization in the genetic code origin as the number of codified amino acids increases.

We have assumed that the coevolution theory of genetic code origin (Wong JT, Proc Natl Acad Sci USA 72:1909-1912, 1975) is essentially correct. This theory makes it possible to identify at least 10 evolutionary stages through which genetic code organization might have passed prior to reaching its current form. The calculation of the minimization level of all these evolutionary stages leads to the following conclusions. (1) The minimization percentages increased linearly with the number of amino acids codified in the codes of the various evolutionary stages when only the sense changes are considered in the analysis. This seems to favor the physicochemical theory of genetic code origin even if, as discussed in the paper, this observation is also compatible with the coevolution theory. (2) For the first seven evolutionary stages of the genetic code, this trend is less clear and indeed is inverted when we consider the global optimisation of the codes due to both sense changes and synonymous changes. This inverse correlation between minimization percentages and the number of amino acids codified in the codes of the intermediate stages seems to favor neither the physicochemical nor the stereochemical theories of genetic code origin, as it is in the early and intermediate stages of code development that these theories would expect minimization to have played a crucial role, and this does not seem to be the case. However, these results are in agreement with the coevolution theory, which attributes a role to the physicochemical properties of amino acids that, while important, is nevertheless subordinate to the mechanism which concedes codons from the precursor amino acids to the product amino acids as the primary factor determining the evolutionary structuring of the genetic code. The results are therefore discussed in the context of the various theories proposed to explain genetic code origin.

Algorithms↗
PubMed1995

Binding and substrate specificities of a Streptomyces olivaceoviridis chitinase in comparison with its proteolytically processed form.

Streptomyces olivaceoviridis is an efficient chitin degrader. One of its genes encoding an exochitinase (exo-ChiO1) was previously characterized. The transcription was found to be inducible by chitin, but not by glucose. The transcriptional start site is situated 38 bp upstream of the start codon. S. olivaceoviridis as well as transformants of S. vinaceus and S. lividans carrying the exo-chiO1 gene on a multicopy vector secrete a 59-kDa chitinase which adheres strongly and under most conditions irreversibly to the substrate chitin. After having released the enzyme from the crystalline substrate in the presence of high concentrations of guanidine hydrochloride, it was purified to homogeneity by consecutive chitin- and immunoaffinity chromatographies. Immunofluorescence microscopy revealed that the enzyme specifically binds to crystalline alpha-chitin within fungi and other organisms as well as to beta-chitin, but not to colloidal chitin, chitosan, various types of cellulose, or other polysaccharides. The amino acids deduced from the highly specific binding domain (12 kDa) of this enzyme do not share significant similarity with any known region interacting with chitin or another substrate. During cultivation with chitin, the 59-kDa enzyme is proteolytically processed to a 47-kDa truncated chitinase lacking the chitin-binding domain. The 47-kDa enzyme hydrolyses crystalline chitin considerably less efficiently than the 59-kDa enzyme, whereas colloidal chitin and low-molecular-mass substrates are quite equally degraded by both enzymes at identical optimal pH (7.3) and temperature (45-55 degrees C) values. Thus a strong adhesion of the enzyme to its crystalline substrate via its binding domain is a prerequisite for efficient hydrolysis.

Base Sequence↗
PubMed2002

Limitations of relative apparent synapomorphy analysis (RASA) for measuring phylogenetic signal.

In this paper we use hypothetical and empirical data matrices to evaluate the ability of relative apparent synapomorphy analysis (RASA) to measure phylogenetic signal, select outgroups, and identify terminals subject to long-branch attraction. In all cases, except for equal character-state frequencies, RASA indicated extraordinarily high levels of phylogenetic information for hypothetical data matrices that are uninformative regarding relationships among the terminals. Yet, regardless of the number of characters or character-state frequencies, RASA failed to detect phylogenetic signal for hypothetical matrices with strong phylogenetic signal. In our empirical example, RASA indicated increasing phylogenetic signal for matrices for which the strict consensus of the most parsimonious trees is increasingly poorly resolved, clades are increasingly poorly supported, and for which many relationships are in conflict with more widely sampled analyses. RASA is an ineffective approach to identify outgroup terminal(s) with the most plesiomorphic character states for the ingroup. Our hypothetical example demonstrated that RASA preferred outgroup terminals with increasing numbers of convergent character states with ingroup terminals, and rejected the outgroup terminal with all plesiomorphic character states. Our empirical example demonstrated that RASA, in all three cases examined, selected an ingroup terminal, rather than an outgroup terminal, as the best outgroup. In no case was one of the two outgroup terminals even close to being considered the optimal outgroup by RASA. RASA is an ineffective means of identifying problematic long-branch terminals. In our hypothetical example, RASA indicated a terminal as being a problematic long-branch terminal in spite of the terminal being on a zero-length branch and having no possibility of undergoing long-branch attraction with another terminal. RASA also failed to identify actual problematic long-branch terminals that did undergo long-branch attraction, but only after following Lyons-Weiler and Hoelzer's (1997) three-step process to identify and remove terminals subject to long-branch attraction. We conclude that RASA should not be used for any of these purposes.

Algorithms↗
PubMed1995

Analysis of antigenic surfaces of proteins.

The processes underlying molecular evolution have proved difficult to understand due to the complexity and obscurity of the selective pressures at work. The strong selective pressure to optimize antigen recognition means that antibody paratopes are more favorable systems than most in which to investigate these processes, as are viral epitopes, which evolve under an inverse pressure, rapidly changing to avoid recognition. Because recognition is a property of the surfaces of these molecules we expect that their evolutionary development may be read in the changes in the 3-dimensional array of chemical groups displayed on their surface. We have analyzed the bulk properties of these surfaces and find that there are significant differences in exposed amino acid preferences among 1) a control group of immunologically secluded proteins, 2) binding surfaces of immunoglobulins, and 3) the outer surfaces of picornaviruses. Compared to the control group, the immunoglobulin complementarity determining regions possess a relative excess of serine residues whereas picornaviruses suppress serine but overuse threonine residues, suggesting that the differing selective pressure has led to perturbations in the population of amino acid types on the surface of these proteins. Although these changes may be rationalized in terms of the structure and chemistry of the different side chains, we suggest that there may be a further, genetic component behind the observations: point mutations in the respective codons for serine and threonine lead to markedly different forms of structural variability. The high rate of reselection observed for these residues is in line with this second mechanism.

Amino Acids↗
PubMed1993

High-level expression of the Japanese encephalitis virus E protein by recombinant vaccinia virus and enhancement of its extracellular release by the NS3 gene product.

Recombinant vaccinia viruses expressing the prM and E genes of the Japanese encephalitis virus (JEV) were constructed by use of synthetic promoters. While the recombinant virus mOJ6-SL, with an optimized vaccinia late-gene promoter, produced a 20-fold elevated level of E protein, as well as an 86-kDa precursor protein in infected cells, no significant quantitative difference was detected between the extracellular or cell-surface E protein produced by mOJ6-SL and those produced by mOJ6 with the 7.5-kDa promoter. However, when the cells were infected with Dengue 2 virus before infection with mOJ6-SL, the amount of the extracellular E protein increased 16-fold. In addition, enhancement of its extracellular release was observed when cells were co-infected with mOJ6-SL and recombinant vaccinia virus expressing the NS3 gene of JEV.

Amino Acid Sequence↗
PubMed1996

Efficiencies of different genes and different tree-building methods in recovering a known vertebrate phylogeny.

The relative efficiencies of different protein-coding genes of the mitochondrial genome and different tree-building methods in recovering a known vertebrate phylogeny (two whale species, cow, rat, mouse, opossum, chicken, frog, and three bony fish species) was evaluated. The tree-building methods examined were the neighbor joining (NJ), minimum evolution (ME), maximum parsimony (MP), and maximum likelihood (ML), and both nucleotide sequences and deduced amino acid sequences were analyzed. Generally speaking, amino acid sequences were better than nucleotide sequences in obtaining the true tree (topology) or trees close to the true tree. However, when only first and second codon positions data were used, nucleotide sequences produced reasonably good trees. Among the 13 genes examined, Nd5 produced the true tree in all tree-building methods or algorithms for both amino acid and nucleotide sequence data. Genes Cytb and Nd4 also produced the correct tree in most tree-building algorithms when amino acid sequence data were used. By contrast, Co2, Nd1, and Nd41 showed a poor performance. In general, large genes produced better results, and when the entire set of genes was used, all tree-building methods generated the true tree. In each tree-building method, several distance measures or algorithms were used, but all these distance measures or algorithms produced essentially the same results. The ME method, in which many different topologies are examined, was no better than the NJ method, which generates a single final tree. Similarly, an ML method, in which many topologies are examined, was no better than the ML star decomposition algorithm that generates a single final tree. In ML the best substitution model chosen by using the Akaike information criterion produced no better results than simpler substitution models. These results question the utility of the currently used optimization principles in phylogenetic construction. Relatively simple methods such as the NJ and ML star decomposition algorithms seem to produce as good results as those obtained by more sophisticated methods. The efficiencies of the NJ, ME, MP, and ML methods in obtaining the correct tree were nearly the same when amino acid sequence data were used. The most important factor in constructing reliable phylogenetic trees seems to be the number of amino acids or nucleotides used.

Algorithms↗
PubMed1993

Characterization of ribonuclease H activities present in two cell-free protein synthesizing systems, the wheat germ extract and the rabbit reticulocyte lysate.

Experimental evidence accumulated to date by several research groups indicates that antisense oligodeoxynucleotides targeted against messenger RNA (mRNA) sequences located downstream of the initiation codon fail to inhibit the translation of this mRNA unless the hybrid is cleaved by RNase H. It has previously been shown that exogenous RNase H has to be added to rabbit reticulocyte lysate to obtain translational arrest (unless freshly prepared lysates are used). In contrast there is no need of exogenous RNase H by using wheat germ extract for translation because the level of endogenous RNase H is high enough to ensure cleavage of the hybrid formed between the antisense oligodeoxyribonucleotide and its complementary sequence on the mRNA. Surprisingly, we found that these two cell-free translation systems display similar amounts of RNase H activities when tested under standard conditions (extract diluted 500 times in the RNase H reaction mix). The RNase H activity of the rabbit reticulocyte lysate has a divalent cation requirement and sensitivity to inhibitors similar to class I ribonuclease H, whereas the activity of the wheat germ extract shows similarities to class II ribonuclease H. However, when these activities were assayed under conditions similar to those used for translation experiments, only highly reduced levels of activity were found in comparison to the standard assays. This reduction is due in part to sub-optimal ionic conditions for the endogenous RNase H activities in these extracts, and, for the other part, likely due to interactions with other proteins present in the lysates. In these conditions, however, the remaining activity found in the wheat germ extract was three times higher than the activity found in the rabbit reticulocyte lysate. Whether this difference can by itself explain the indicated differences in the two systems observed in hybrid-arrest of translation experiments remains open to discussion.

Animals↗
PubMed2006

Dynamic evolution of selenocysteine utilization in bacteria: a balance between selenoprotein loss and evolution of selenocysteine from redox active cysteine residues.

BACKGROUND: Selenocysteine (Sec) is co-translationally inserted into protein in response to UGA codons. It occurs in oxidoreductase active sites and often is catalytically superior to cysteine (Cys). However, Sec is used very selectively in proteins and organisms. The wide distribution of Sec and its restricted use have not been explained. RESULTS: We conducted comparative genomics and phylogenetic analyses to examine dynamics of Sec decoding in bacteria at both selenium utilization trait and selenoproteome levels. These searches revealed that 21.5% of sequenced bacteria utilize Sec, their selenoproteomes have 1 to 31 selenoproteins, and selenoprotein-rich organisms are mostly Deltaproteobacteria or Firmicutes/Clostridia. Evolutionary histories of selenoproteins suggest that Cys-to-Sec replacement is a general trend for most selenoproteins. In contrast, only a small number of Sec-to-Cys replacements were detected, and these were mostly restricted to formate dehydrogenase and selenophosphate synthetase families. In addition, specific selenoprotein gene losses were observed in many sister genomes. Thus, the Sec/Cys replacements were mostly unidirectional, and increased utilization of Sec by existing protein families was counterbalanced by loss of selenoprotein genes or entire selenoproteomes. Lateral transfers of the Sec trait were an additional factor, and we describe the first example of selenoprotein gene transfer between archaea and bacteria. Finally, oxygen requirement and optimal growth temperature were identified as environmental factors that correlate with changes in Sec utilization. CONCLUSION: Our data reveal a dynamic balance between selenoprotein origin and loss, and may account for the discrepancy between catalytic advantages provided by Sec and the observed low number of selenoprotein families and Sec-utilizing organisms.

Bacteria↗
PubMed1993

A two-dimensional model at the nucleotide level for the central hairpin of coliphage Q beta RNA.

We describe the construction and testing of a structural model at the nucleotide level for conformation CH of the central hairpin of genomic RNA from coliphage Q beta. The model was developed with the computer program MFOLD using both optimal and suboptimal predictions. Structural information obtained by electron microscopic analysis of Kleinschmidt spreadings of Q beta RNA was used to guide the modeling. The model was tested in solution with three enzymatic probes: RNase T1, RNase T2, and RNase V1, as well as four chemical probes: dimethylsulfate, diethylpyrocarbonate, kethoxal and 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluene sulfonate (CMCT). The structural analyses in solution are consistent with the predicted structural model. The model is also supported by comparative structural analysis with the related coliphage SP. The model provides a structural basis for published biochemical and genetic studies implicating large, long-range structural features in the co-regulation of viral coat and replicase expression. In addition, we show that the read-through region of the viral protein A1 forms a separate structural domain, and we suggest that it functions as a nucleation site that participates in the folding and refolding of the molecule during replication and translation. In addition to the central hairpin, we have analyzed the structure of the viral coat initiation region. Our studies show that the entire region consists of small local hairpins and that 26 nucleotides immediately surrounding the coat initiation codon are single-stranded.

Base Sequence↗