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PubMed1999

A point mutation in a plant calmodulin is responsible for its inhibition of nitric-oxide synthase.

The calcium/calmodulin-dependent activation of nitric-oxide synthase (NOS) and its production of nitric oxide (NO) play a key regulatory role in plant and animal cell function. SCaM-1 is a plant calmodulin (CaM) isoform that is 91% identical to mammalian CaM (wild type CaM (wtCaM)) and a selective competitive antagonist of NOS (Cho, M. J., Vaghy, P. L., Kondo, R., Lee, S. H., Davis, J. P., Rehl, R., Heo, W. D., and Johnson, J. D. (1998) Biochemistry 37, 15593-15597). We have used site-directed mutagenesis to show that a point mutation, involving the substitution of valine for methionine at position 144, is responsible for SCaM-1's inhibition of mammalian NOS. An M144V mutation in wild type CaM produced a mutant (M144V) which exhibited nearly identical inhibition of NOS's NO production and NADPH oxidation, with a similar K(i) (approximately 15 nM) as SCaM-1. A V144M back mutation in SCaM-1 significantly restored its ability to activate NOS's catalytic functions. The length of the hydrophobic amino acid side chain at position 144 appears to be critical for NOS activation, since M144L and M144F activated NOS while M144V and M144C did not. Despite their competitive antagonism of NOS, M144V, like SCaM-1, exhibited a similar dose-dependent activation of phosphodiesterase and calcineurin as wtCaM. SCaM-1 and M144V produced greater inhibition of NOS's oxygenase domain function (NO production) than its reductase domain functions (NADPH oxidation and cytochrome c reduction). Thus, CaM's methionine 144 plays a critical role the activation of NOS, presumably by influencing the function of NOS's oxygenase domain.

Calmodulin↗
PubMed1994

A point mutation in the bile acid biosynthetic enzyme sterol 27-hydroxylase in a family with cerebrotendinous xanthomatosis.

Cerebrotendinous xanthomatosis (CTX) is a rare familial disorder characterized by progressive neurological dysfunction, atherosclerosis, and xanthomas with sterol storage in the nervous system, vessels, and tendons. Increased serum cholestanol, derived from intermediates of cholesterol catabolism, may possibly be a major cause of the disease. An examination was made of the cDNA encoding cytochrome P450 sterol 27-hydroxylase (CYP27) in hepatic mitochondria, considered a defective enzyme inducing CTX, in a Japanese housewife afflicted with CTX and her family. The proposita and one of her brothers, who also had CTX symptoms and hypercholestanolemia, were found to be homozygotic, carrying a point mutation in the CYP27 gene at Arg104 (CGG) to Trp104 (TGG). The mutant position has a 100% conserved positive charge in all known vertebrate cytochrome P450s and even in bacterial cytochrome P450cam. The mother of the proposita and another brother were both free of CTX symptoms and were heterozygotic for the mutation, although their plasma cholesterol increased moderately. An increase in plasma cholestanol alone would, thus, not appear to be a direct cause of sterol storage in CTX, while CTX is strongly suggested to be caused by defects in both alleles of the CYP27 gene.

Aged↗
PubMed2000

A single point mutation in TFIIA suppresses NC2 requirement in vivo.

Negative cofactor 2 (NC2) is a dimeric histone-fold complex that represses RNA polymerase II transcription through binding to TATA-box-binding protein (TBP) and inhibition of the general transcription factors TFIIA and TFIIB. Here we study molecular mechanisms of repression by human NC2 in vivo in yeast. Yeast NC2 genes are essential and can be exchanged with human NC2. The physiologically relevant regions of NC2 have been determined and shown to match the histone-fold dimerization motif. A suppressor screen based upon limiting concentrations of NC2beta yielded a cold-sensitive mutant in the yeast TFIIA subunit Toa1. The single point mutation in Toa1 alleviates the requirement for both subunits of NC2. Biochemical characterization indicated that mutant (mt)-Toa1 dimerizes well with Toa2; it supports specific recognition of the TATA box by TBP but forms less stable TBP-TFIIA-DNA complexes. Wild-type but not the mt-Toa1 can relieve NC2 effects in purified transcription systems. These data provide evidence for a dimeric NC2 complex that is in an equilibrium with TFIIA after the initial binding of TBP to promoter TATA boxes.

Base Sequence↗
PubMed1993

Transcript identification in the optomotor-blind locus of Drosophila melanogaster by intragenic recombination mapping and PCR-aided sequence analysis of lethal point mutations.

The optomotor-blind gene of Drosophila melanogaster is large and genetically complex. Five partly independent complementation groups are uncovered by several viable and lethal mutations at the locus. At least 15 RNA signals have been detected by Northern blot analysis. One of them, T3, derived from a 75 kb primary transcript, has been proposed as the carrier of optomotor-blind function, based on the large size of its precursor and its tissue distribution. We here provide direct evidence that T3 is the optomotor-blind transcript. A facile and generally applicable selection scheme for the isolation of intragenic meiotic recombinants was applied to map two lethal optomotor-blind point mutations to exons of the T3 transcript. Amplification of mutant DNA by the polymerase chain reaction (PCR) and sequencing of the amplified exons revealed the presence of mutations that lead to truncation of the T3 open reading frame. The recombination rate observed in the optomoter-blind locus is within the range of rates that have been determined in a few other Drosophila loci.

Amino Acid Sequence↗
PubMed1994

Conversion of plasminogen activator inhibitor-1 from inhibitor to substrate by point mutations in the reactive-site loop.

Plasminogen activator inhibitor-1 (PAI-1), the main physiological inhibitor of tissue-type plasminogen activator (t-PA), may occur in three interconvertible conformations: active, latent, and substrate. To delineate specific domains in the PAI-1 molecule responsible for its conformational flexibility and associated functional diversity, four mutants of PAI-1 (with the amino acids at positions P12, P10, P8, and P6, respectively, substituted with proline) were expressed in Escherichia coli, purified, and characterized. Wild-type PAI-1 (wtPAI-1) had a specific activity of 21 +/- 10% (mean +/- S.D., n = 3) of the theoretical maximum value. PAI-1-P12 (Ala-->Pro at P12), PAI-1-P10 (Ser-->Pro at P10), and PAI-1-P8 (Thr-->Pro at P8) had specific activities of 0.06 +/- 0.03% (n = 3), 2.6 +/- 1.0% (n = 4), and 2.7 +/- 1.1% (n = 3), respectively (p < 0.03 versus wtPAI-1). PAI-1-P6 (Val-->Pro at P6) has a specific activity of 12 +/- 3.3% (n = 3) of the theoretical maximum value (p = not significant versus wtPAI-1). SDS-polyacrylamide gel electrophoresis of mixtures of wtPAI-1 or PAI-1-P6 with a 2-fold molar excess of t-PA yielded a mixture of a covalent 110-kDa t-PA.PAI-1 complex (15-25%), nonreactive 45-kDa material (44-67%), and a 41-kDa band (18-31%) representing cleaved PAI-1. PAI-1-P12, PAI-1-P10, and PAI-1-P8 behaved as substrates, yielding predominantly the 41-kDa cleavage product (85-91%) and a small amount (9-15%) of non-reactive material. NH2-terminal amino acid sequencing revealed that cleavage occurred at the P1-P1' bond (Arg346-Met347). Incubation of PAI-1-P12, PAI-1-P10, or PAI-1-P8 with a 2-fold molar excess of urokinase-type plasminogen activator, plasmin, or thrombin also primarily generated a 41-kDa cleavage product (62-89%). Incubation of wtPAI-1 and PAI-1-P6 at 37 degrees C resulted in a loss of inhibitory activity, whereas the substrate behavior of PAI-1-P12, PAI-1-P10, and PAI-1-P8 remained unaltered. Treatment of the three substrate-like mutants with guanidinium Cl did not induce inhibitory activity. In conclusion, point mutations at positions P12, P10, and P8 yield PAI-1 variants with stable substrate properties, which may facilitate more detailed structure/function studies.

Amino Acid Sequence↗
PubMed1993

Investigation of the role of the ras protooncogene point mutation in human uveal melanomas.

PURPOSE: Genetic alterations have been observed in a wide variety of neoplastic processes, including Burkitt's lymphoma, chronic myelogenous leukemia, promyelocytic leukemia, and solid tumors of the colon, skin, and breast. The polymerase chain reaction (PCR), dot blotting, and direct double-stranded DNA sequencing were used to assess ras gene activation in human uveal melanomas for three candidate genes: c-Ha-ras1, c-Ki-ras2, and N-ras at codons 12, 13, and 61. METHODS: Samples of 49 human uveal melanomas were obtained. Amplifiable high molecular weight DNA was obtained from 39 of these. PCR amplification of regions centering on three candidate ras genes was performed. PCR-amplified DNA was evaluated by dot blot and double-stranded DNA sequencing utilizing standard methods. RESULTS: No point mutations were identified in screening the c-Ha-ras gene nor were any genetic alterations found in the c-Ki-ras2 gene at codons 12 and 13. Only wild-type sequences were found at codon 61. No ras mutations were detected in any uveal melanomas studied. CONCLUSIONS: This study provides no evidence to support an association between ras protooncogene mutations and human uveal melanomas at codons 12, 13, or 61.

Adult↗
PubMed2003

A novel c-kit positive biphenotypic acute leukemia cell line, TMBL-1, carrying a p53 point mutation.

We established and characterized a c-kit positive cell line from the bone marrow of a patient with biphenotypic acute leukemia (BAL). The cell line, designated TMBL-1, carried a His-175 mutant p53. The immunophenotype of the primary leukemia cells at diagnosis was cytoplasmic CD3+, CD7+, CD13+, CD33-, interleukin-7 (IL-7) receptor+ and c-kit -. However, leukemia cells in relapse and TMBL-1 cells were CD33+ and c-kit +. Immunophenotypically, TMBL-1 is a BAL cell line that coexpresses T-lymphoid and myeloid markers which fulfill the criteria of the European Group for the Immunological Characterization of Leukemia. Stem cell factor (SCF), a key regulator of hematopoiesis signaling through c-kit, enhanced the proliferation of TMBL-1 cells. Direct sequencing revealed the conversion at codon 175 of the p53 gene in the TMBL-1 cells. Primary leukemia cells in relapse also carried the same point mutation but not at diagnosis. Moreover, TMBL-1 cells are sensitive to paclitaxel, which could induce p53-independent apoptosis. The biphenotypic features and p53 mutation may be associated with progression to a more malignant type. This cell line may provide new information on the role of SCF in the overlapping area between early T-lymphoid/myeloid cells, and help in the design of new therapies targeted towards p53 mutations.

Adult↗
PubMed2006

Synthesis and conformational analysis of Id2 protein fragments: impact of chain length and point mutations on the structural HLH motif.

The Id proteins are negative regulators of several basic-helix-loop-helix (HLH) transcription factors, including the ubiquitous E factors and the tissue-specific myogenin-regulating factors. Id1 through Id4 contain highly identical HLH domains but different N- and C-terminal extensions. Beside the heterodimerization with the parent HLH factors, Id2 was shown to additionally interact with the retinoblastoma protein and to be overexpressed in neuroblastoma. Thus, Id2 represents an interesting target for cancer therapy based on the inhibition of protein-protein interactions. Here we present the synthesis and circular dichroism (CD) analysis of peptides derived from point mutations and N-/C-terminal truncations of Id2. The helix character of the HLH domain (residues 36-76) was reduced upon substitution of Met39/-62 and Cys42 with Nle and Ser, respectively, suggesting a structural role of these side chains. The largest sequence that could be obtained by stepwise solid-phase peptide synthesis (SPPS) with Fmoc strategy spanned the entire HLH motif (with Cys42 replaced by Ser) and part of the C-terminus (residues 77-110). This 75-residue long fragment was less helical than the isolated HLH domain and had propensity to aggregate, which was correlated with the presence of the flanking residues C-terminal to helix-2. By CD analysis of an equimolar mixture of the sequence 36-110 with the N-terminus 1-35, noncovalent interactions between the two peptides were detected, which, however, changed upon aging. In contrast, the mixture of the HLH sequence 36-76 with the N-terminus was characterized by a stabilized helix structure that was maintained also upon aging. Presumably, the N-terminal region interacted with the folded HLH motif in a specific manner, whereas only unspecific, weak contacts occurred with the partly unfolded HLH domain and/or the immediate flanking residues 77-110.

Amino Acid Sequence↗
PubMed1998

A point mutation (D79N) of the alpha2A adrenergic receptor abolishes the antiepileptogenic action of endogenous norepinephrine.

Norepinephrine serves as a neurotransmitter for a population of neurons the cell bodies of which reside in a brainstem nucleus and the axons of which project widely to discrete subsets of forebrain neurons. Norepinephrine powerfully inhibits epileptogenesis in the kindling model. Pharmacological methods have demonstrated that the antiepileptogenic actions of norepinephrine are exerted via alpha2 adrenergic receptors residing on targets of noradrenergic neurons. The existence of three alpha2 adrenergic receptor subtypes together with the lack of subtype-specific ligands has precluded understanding the role of individual alpha2 adrenergic receptor subtypes in the antiepileptogenic actions of norepinephrine. Gene targeting was used to introduce a point mutation into the alpha2A adrenergic subtype in the mouse genome. The mutation produced a marked enhancement of epileptogenesis and abolished the proepileptogenic actions of the alpha2 adrenergic receptor antagonist idazoxan. These studies reveal the crucial contribution of the alpha2A receptor subtype in suppression of epileptogenesis. Development of agents that promote selective activation of the alpha2A receptor subtype may provide novel therapeutic strategies for the prophylaxis of epilepsy.

Adrenergic alpha-Agonists↗
PubMed1998

Novel point mutations in the alphaIIb subunit (Phe289-->Ser, Glu324-->Lys and Gln747-->Pro) causing thrombasthenic phenotypes in four Japanese patients.

We analysed the molecular basis of Glanzmann thrombasthenia (GT) in four Japanese patients with type I or type II disease. Polymerase chain reaction (PCR) and subsequent direct sequencing of platelet RNA and genomic DNA revealed three single nucleotide substitutions of the alphaIIb gene, which were confirmed by allele-specific PCR or restriction analysis. One patient with type I GT had a T to C base substitution in exon 11 resulting in a Phe (TTT)-289 to Ser (TCT) mutation (F289S) of the subunit. Another type I patient had a G to A base substitution in exon 12 resulting in a Glu (GAA)-324 to Lys (AAA) mutation (E324K). Interestingly, two unrelated patients with type II GT shared an A to C base substitution in exon 2 3, a region previously not associated with GT, resulting in a Gln (CAA)-747 to Pro (CCA) mutation (Q747P). To analyse the effects of these mutations on alphaII(b)beta3 surface expression, the wild-type alphaIIb cDNA or mutant alphaIIb cDNAs were transfected into Chinese hamster ovary (CHO) cells together with a wild-type beta3 cDNA. Flow cytometric analysis using an anti-alphaII(b)beta3 complex antibody revealed that 50.6% of CHO cells with wild-type alphaII(b)beta3 expressed complexes, whereas only 1 6%, 7.7% and 31.3% of cells, with IIb(F289S)beta3, alphaIIb(E324K)beta3 and alphaIIb(Q747P)beta3 expressed complexes, respectively. Our data indicate that these three novel point mutations in the alphaIIb subunit may hamper surface expression of the alphaII(b)beta3 complex, thus resulting in the quantitative GT phenotypes of platelets from these patients.

Adult↗
PubMed1992

Retinoic acid-resistant HL-60R cells harbor a point mutation in the retinoic acid receptor ligand-binding domain that confers dominant negative activity.

Retinoic acid (RA) induces granulocytic differentiation of acute promyelocytic leukemia (APL) cells and is a useful therapeutic agent for patients with this disease. In the HL-60 promyelocytic leukemia cell line, this RA-induced granulocytic differentiation appears to be directly mediated through the RA receptor (RAR-alpha). We have previously identified a mutant subclone of HL-60 (designated HL-60R) that exhibits relative resistance to RA and that harbors RA receptors with markedly reduced affinity for RA. In the present study, we have now identified the genetic basis for this aberrant RA receptor activity. DNA sequencing of polymerase chain reaction-amplified cDNA products corresponding to the RAR-alpha ligand-binding domain shows a point mutation in RAR-alpha codon 411 in this mutant HL-60R subclone. This specific C-->T mutation generates a termination codon resulting in the truncation of 52 amino acids at the COOH terminal end of RAR-alpha. In cotransfection studies, expression vectors harboring this mutated RAR-alpha exhibit dominant negative activity with respect to the trans-activating function of the normal RAR-alpha. Although our observations are limited to HL-60 cells, similar RA receptor mutations might play an important role in the acquisition of RA resistance in RA-treated APL patients.

Amino Acid Sequence↗
PubMed1999

Conformational studies of parathyroid hormone (PTH)/PTH-related protein (PTHrP) point-mutated hybrids.

The N-terminal 1-34 segments of both parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP) bind and activate the same membrane receptor in spite of major differences between the two hormones in their amino acid sequence. Recently, it was shown that in (1-34)PTH/PTHrP segmental hybrid peptides, the N-terminal 1-14 segment of PTHrP is incompatible with the C-terminal 15-34 region of PTH leading to substantial reduction in potency. The sites of incompatibility were identified as positions 5 in PTH and 19 in PTHrP. In the present paper we describe the synthesis, biological evaluation, and conformational characterization of two point-mutated PTH/PTHrP 1-34 hybrids in which the arginine residues at positions 19 and 21 of the native sequence of PTHrP have been replaced by valine (hybrid V(21)) and glutamic acid (hybrid E(19)), respectively, taken from the PTH sequence. Hybrid V(21) exhibits both high receptor affinity and biological potency, while hybrid E(19) binds weakly and is poorly active. The conformational properties of the two hybrids were studied in aqueous solution containing dodecylphosphocholine (DPC) micelles and in water/2,2, 2-trifluoroethanol (TFE) mixtures. Upon addition of TFE or DPC micelles to the aqueous solution, both hybrids undergo a coil-helix transition. The maximum helix content in 1 : 1 water/TFE, obtained by CD data for both hybrids, is approximately 80%. In the presence of DPC micelles, the maximum helix content is approximately 40%. The conformational properties of the two hybrids in the micellar system were further investigated by combined 2D-nmr, distance geometry (DG), and molecular dynamics (MD) calculations. The common structural motif, consisting of two helical segments located at N- and C-termini, was observed in both hybrids. However, the biologically potent hybrid V(21) exhibits two flexible sites, centered at residues 12 and 19 and connecting helical segments, while the flexibility sites in the weakly active hybrid E(19) are located at position 11 and in the sequence 20-26. Our findings support the hypothesis that the presence and location of flexibility points between helical segments are essential for enabling the active analogs to fold into the bioactive conformation upon interaction with the receptor.

Cell Line↗
PubMed1994

Point mutation of aldehyde dehydrogenase-2 gene in mutant strains of Long-Evans rats.

Long-Evans Cinnamon (LEC) and Long-Evans Agouti (LEA) rats are mutant strains established from Long-Evans rats. LEC rats display hereditary hepatitis and spontaneous hepatocellular carcinoma, but LEA rats do not develop liver diseases. We previously demonstrated that LEC rats had an impairment of liver aldehyde dehydrogenase (ALDH) activities, and all LEC rats which were fed with a liquid diet containing 5% ethanol died within 2 weeks. In the present study, we also found that LEA rats could not metabolize ethanol and died after being fed the same diet. Remarkably, in the liver of LEA rats, low Km ALDH activities were suppressed as much as in LEC rats. These results suggested that both LEC and LEA rats have hereditary deficiencies in ALDH. Nucleotide sequence analysis of ALDH2 genes in both LEC and LEA rats demonstrated that the point mutation of the codon for residue 67 encoding Gln to Asp was observed; this was not so in either Long-Evans rats or Wistar rats. This mutation in ALDH2 genes may cause inactivation of ALDR activity in LEC and LEA rats.

Aldehyde Dehydrogenase↗
PubMed2003

Identification of a new point mutation in the human molybdenum cofactor sulferase gene that is responsible for xanthinuria type II.

A 43-year-old xanthinuric female was referred to our department because of hypouricemia. Routine laboratory data showed hypouricemia, a high level of plasma oxypurines, decreased urinary uric acid excretion, and increased urinary oxypurine excretion, with xanthine dehydrogenase activity in the duodenal mucosa below the limits of detection. In addition, allopurinol was not metabolized. From these findings, the patient was diagnosed with xanthinuria type II. To investigate the properties of xanthine dehydrogenase/xanthine oxidase (XDH/XO) deficiency, a cDNA sequence encoding XDH/XO, aldehyde oxidase (AO), and molybdenum cofactor sulferase (MCS), as well as immunoblotting analysis for XDH/XO protein, obtained from duodenal mucosa samples were performed. The XDH/XO cDNA and AO cDNA sequences of the xanthinuric patient were consistent with previously reported ones, whereas the MCS cDNA sequence revealed a point mutation of G to C in nucleotide 466, which changed codon 156 from GCC (Ala) to CCC (Pro). In addition, the MCS genomic DNA sequence including the site of the mutation revealed the same, suggesting that the xanthinuric patient was homozygous for this mutation. Such findings have not been previously reported for patients with xanthinuria type II.

Adult↗
PubMed2004

Characterization of a point mutation in the parC gene of Mycoplasma bovirhinis associated with fluoroquinolone resistance.

Quinolone-resistant (QR) mutants of Mycoplasma bovirhinis strain PG43 (type strain) were generated by stepwise selection in increasing concentrations of enrofloxacin (ENR). An alteration was found in the quinolone resistance-determining region (QRDR) of the parC gene coding for the ParC subunit of topoisomerase IV from these mutants, but not in the gyrA, gyrB, and parE gene coding for the GyrA and GyrB subunits of DNA gyrase and the ParE subunit of topoisomerase IV. Similarly, such an alteration in QRDR of parC was found in the field isolates of M. bovirhinis, which possessed various levels of QR. The substitution of leucine (Leu) by serine (Ser) at position 80 of QRDR of ParC was observed in both QR-mutants and QR-isolates. This is the first report of QR based on a point mutation of the parC gene in M. bovirhinis.

Animals↗
PubMed1998

A point mutation in an intronic branch site results in aberrant splicing of COL5A1 and in Ehlers-Danlos syndrome type II in two British families.

Ehlers-Danlos syndrome (EDS) is a heterogeneous group of connective-tissue disorders characterized by skin fragility, joint laxity, and skeletal deformities. Type V collagen appears to have a causal role in EDS types I and II, which show phenotypic overlap and may sometimes be allelic. Type V collagen can exist as a heterotrimer, [alpha1(V)]2alpha2(V), and it both coassembles with and regulates type I collagen-fibril diameter. Using an intragenic COL5A1 polymorphism, we have demonstrated linkage, at zero recombination, to the same allele in two large British EDS type II families (LOD scores 4.1 and 4.3). Affected members from each family were heterozygous for a point mutation in intron 32 (IVS32:T-25G), causing the 45-bp exon 33 to be lost from the mRNA in approximately 60% of transcripts from the mutant gene. This mutation lies only 2 bp upstream of a highly conserved adenosine in the consensus branch-site sequence, which is required for lariat formation. Although both families shared the same marker allele, we have been unable to identify a common genealogy. This is the first description of a mutation at the lariat branch site, which plays a pivotal role in the splicing mechanism, in a collagen gene. Very probably, the resulting in-frame exon skip has a dominant-negative effect due to incorporation of the mutant proalpha chain into the triple-helical molecule. These findings further confirm the importance of type V collagen in the causation of EDS type II, and the novel collagen mutation indicates the importance of the lariat branch site in splicing.

Alternative Splicing↗
PubMed2002

Modulation of homo- and heterodimerization of Harvey sarcoma virus RNA by GACG tetraloops and point mutations in palindromic sequences.

Retroviruses harbour a diploid genome of two plus-strand RNAs linked non-covalently at the dimer linkage structure. Co-packaging of two parental RNAs is a prerequisite for recombination in retroviruses, but formation of heterodimers has not been demonstrated directly in vivo. Here, we explore elements in Harvey sarcoma virus (HaSV) RNA involved in homodimerization and heterodimerization with RNA of Moloney (Mo) and Akv murine leukemia viruses (MLV). By an in vitro assay, we found that HaSV dimerization specificity could be modulated by mutations in a decanucleotide palindrome (Pal) probably folded into a kissing-loop. Autocomplementary and non-autocomplementary sequences introduced into the putative loop directed the specificity towards formation of homodimers and heterodimers, respectively. Two stem-loop (SL) structures, both exposing a GACG tetraloop, enhanced the formation of stable HaSV dimers.A similar decanucleotide palindrome has been implicated in homodimerization of MLVs. Heterodimers between HaSV RNA and Mo- or Akv MLV were unstable, but could be stabilized by introduction of two point mutations in the putative HaSV kissing-loop, creating exact complementarity with Mo/Akv MLV palindromes. Moreover, such changes increased the HaSV RNA affinity for the two MLV RNAs. Similar to HaSV RNA homodimers, formation of heterodimers with Mo- or Akv MLV RNAs was induced by the presence of GACG loops. On the basis of these results, we propose that palindromic sequences act as variable determinants of specificity and GACG tetraloops as conserved determinants in the formation of homodimers and heterodimers of gamma-retrovirus retroviral RNAs in vivo. The complementarity of loop sequences in the packaging signal upstream of the GACG tetraloops might therefore determine homo- and heterodimerization specificity and recombination activity of these viruses.

Base Pairing↗
PubMed2005

High-level expression of IMP-5 carbapenemase owing to point mutation in the -35 promoter region of class 1 integron among Pseudomonas aeruginosa clinical isolates.

Eight strains of Pseudomonas aeruginosa producing IMP-5 carbapenemases were collected from three Portuguese hospitals. All isolates were epidemiologically unrelated. The bla(IMP-5) gene was inserted into a class 1 integron previously reported in Acinetobacter baumannii 65FFC. Expression of the the bla(IMP-5) gene in P. aeruginosa has been shown to be driven by the P(1) promoter [TTGATA] in which the cytosine was replaced by thymine, which caused an increase in transcription of bla(IMP-5) that was confirmed by site-directed mutagenesis. The minimum inhibitory concentrations (MICs) of imipenem for Escherichia coli pGMLA-1 (the recombinant from A. baumannii 65FFC) and E. coli pATG-2 (the recombinant from P. aeruginosa isolates) were 0.5 mg/L and >32 mg/L, respectively. This study reports the spread of a class 1 integron In76 with a new point mutation in the P(1) promoter sequence leading to overproduction of IMP-5.

Bacterial Proteins↗