Biomedical subjects

M Syvanen

Publications and source records attributed to M Syvanen.

At least 19 recordsLinked to original sources

PubMed1992

DNA twist as a transcriptional sensor for environmental changes.

A variety of reports describe shifts in the environment which cause a corresponding change in the measured linking number of plasmid DNA isolated from bacterial cells. This change in linking number is often attributed to a change in superhelical density. This, coupled with the observation that transcription is often dependent upon the superhelical density of the DNA template seen in vitro, has led to the suggestion that superhelical density may control expression of certain genes. However, since many environmental changes could, in principle, influence DNA twist itself, then the measured differences in linking number, delta Lk, may simply be a consequence of variation in twist according to the relationship delta Lk = delta Tw + delta Wr, where delta Tw and delta Wr are changes in twist and writhe, respectively. In fact, we show that when an environmental change causes a change in the helical pitch of the DNA, and if the superhelical density of DNA is regulated to remain constant according to the homeostatic model of Menzel and Gellert, then delta Lk approximately delta Tw. We have found that there are a number of published reports describing variation in promoter activity as a function of linking number that can be explained by considering twist. We suggest that there are classes of sigma 70 promoters whose ability to be recognized by RNA polymerase is exquisitely sensitive to the relative orientation of the -35 and -10 regions, and environmental conditions can control this relative orientation by changing DNA twist.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA, Bacterial↗

PubMed1991

Trans-acting transposase mutant from Tn5.

Transposition of Tn5 and of its component insertion sequence IS50R is regulated through the action of two proteins it encodes: a cis-acting transposase, Tnp, and a trans-acting inhibitor of transposition, Inh. The mechanism of the cis-acting Tnp and the relevance of inhibition to cis action have been addressed in the current study. A specific colony morphology assay for transposition of Tn5 was shown to be sensitive to Inh produced in trans and was used to screen for mutants in Inh and/or Tnp with altered regulation. A dominant mutant in IS50R that promotes transposition in trans was isolated and characterized. The mutant (449F) carries a Leu----Phe mutation at position 449 in Tnp. This mutation reduces the frequency of Tn5 or IS50R transposition in cis but allows Tnp-449F to act as efficiently in trans as it does in cis. Tnp-449F is sensitive to inhibition and, furthermore, Inh-449F is a competent inhibitor in trans. These results show that Tnp-449F is a trans-acting transposase, unlike wild-type Tnp, which is cis-acting.

Bacteriophage lambda↗

PubMed1991

Gly-238-Ser substitution changes the substrate specificity of the SHV class A beta-lactamases.

The SHV-type beta-lactamase SHV-2A is related to SHV-1 by a Gly-238-Ser replacement. Strains carrying SHV-2A are resistant to the third generation cephems cefotaxime and ceftizoxime, whereas those that carry SHV-1 are sensitive to these drugs. We present a kinetic analysis of a SHV-1 and SHV-2A enzymes, with the goal of gaining insight into the role of residue 238 in hydrolyzing cefotaxime and ceftizoxime. SHV-2A shows altered kinetic properties for a number of other cephems that also have heterocyclic side chains at the amino position of the 7-aminocephalosporanic acid nucleus (R1 side chain), including a significantly higher kcat/Km than does SHV-1 for cephaloridine, cephalothin, and cefotiam. Two cephems with straight chain R1 substitutions, cephalosporin C and cephacetrile, are not hydrolyzed more efficiently by SHV-2A. These results indicate that the Ser-238-Gly substitution increases the affinity toward cephems with a heterocyclic ring in the R1 side chain. In addition, the data for ampicillin and benzylpenicillin show that addition of a nitrogen to the second carbon of the R1 side chain of a penem results in a lower kcat/Km for SHV-2A relative to SHV-1. These data strongly suggest that the previously proposed hydrogen bond formation between Ser-238 and the second carbon nitrogen of cefotaxime is not an important factor in hydrolysis by SHV-2A. We propose that the Gly-238 to Ser-238 replacement in SHV-2A has altered the hydrophobic pocket so that it can better accommodate cephems with bulky R1 side chains.

Amino Acid Sequence↗

PubMed1991

Molecular cloning of a glutathione S-transferase overproduced in an insecticide-resistant strain of the housefly (Musca domestica).

We report the cloning and sequencing of a glutathione S-transferase (GST) gene from the housefly Musca domestica. A cDNA lambda gt11 library was prepared from the organophosphate insecticide-resistant housefly strain Cornell-R--a variant that has elevated GST activity. The lambda phage GST clone was identified on the basis of its ability to cross-hybridize to a GST DNA probe from Drosophila melanogaster. Based on amino acid homology to other GSTs and expression of GST activity in Escherichia coli, the Musca GST gene (MdGST-1) belongs to the GST gene family. Although organophosphate resistance in Cornell-R is largely due to one of the GSTs, MdGST-1 is probably not the enzyme responsible for resistance. The mutation that controls resistance to organophosphate insecticides in Cornell-R is highly unstable and we isolated spontaneous variants to both insecticide sensitivity and to even higher levels of resistance. This provided us with an isogenic set of three strains. We found that MdGST-1 transcript levels as measured by Northern assays are higher in all three Cornell-R strains relative to the sensitive wild type, but that the sensitive Cornell-R strain has more MdGST-1 transcript than does the highly resistant Cornell-R strain. These data as well as Southern analysis of genomic DNA allow us to conclude: (1) there are multiple GST genes in M. domestica; (2) the natural variant Cornell-R overproduces excess transcript from two and probably more of these genes; and (3) the unstable mutation in Cornell-R influences the levels of multiple GSTs.

Amino Acid Sequence↗

PubMed1991

Evolved neomycin phosphotransferase from an isolate of Klebsiella pneumoniae.

A new aminoglycoside resistance gene (aphA1-IAB) confers high-level resistance to neomycin. The sequence of aphA1-IAB is closely related to aphA1 found in the transposons Tn4352, Tn903 and Tn602. For example, aphA1-IAB differs from aphA1-903 at five nucleotides that result in four amino acid replacements. The enzyme encoded by aphA1-IAB has a significantly higher turnover number with neomycin, kanamycin and G418 as substrates than does the aphA1-903 enzyme. A parsimonious phylogenetic tree suggests that aphA1-IAB evolved from an ancestral form that is closely related or identical to the aphA1 found in Tn903. The excess of replacement substitutions over silent substitutions in aphA1-IAB, as well as its convergence toward aphA3 from Staphylococcus aureus, is indicative of selective evolution. Our hypothesis to explain these results is that aphA1-IAB evolved under the selective pressure of neomycin use in relatively recent times.

Amino Acid Sequence↗

PubMed1990

Contrasting evolutionary histories of chloroplast thioredoxins f and m.

Fourteen thioredoxin sequences were used to construct a minimal phylogenetic tree by using parsimony. The bacterial thioredoxins clustered into three groups: one containing the photosynthetic purple bacteria, Escherichia and Corynebacterium; a second containing the photosynthetic green bacterium, Chlorobium; and a third containing cyanobacteria. These groupings are similar to those generated from earlier 16s RNA analyses. Animal thioredoxins formed a fourth group. The two thioredoxins of chloroplasts (f and m) showed contrasting phylogenetic patterns. As predicted from prior studies, spinach chloroplast thioredoxin m grouped with its counterparts from cyanobacteria and eukaryotic algae, but, unexpectedly, thioredoxin f grouped with the animal thioredoxins. The results indicate that, during evolution, thioredoxin m of contemporary photosynthetic eukaryotic cells was derived from a prokaryotic symbiont, whereas thioredoxin f descended from an ancestral eukaryote common to plants and animals. The findings illustrate the potential of thioredoxin as a phylogenetic marker and suggest a relationship between the animal and f-type thioredoxins.

Amino Acid Sequence↗

PubMed1990

Identification and sequence of the drpA gene from Escherichia coli.

The drpA gene of Escherichia coli encodes a factor that is involved in global RNA synthesis. We establish that the drpA gene has been successfully cloned and describe the fine-structure map of three drpA-(Ts) mutations as well as the complete nucleotide sequence of the drpA gene. We identified a major sigma-70 promoter for the drpA gene on the bases of (i) its similarity to the consensus sequence and (ii) S1 protection and primer extension mapping data. In addition, the nucleotide sequence revealed a pair of dnaA boxes and a factor-independent terminator at the 5' end and 3' end of the gene, respectively. The deduced amino acid sequence of the DrpA protein showed a nucleotide-binding pocket found in some ATPases.

Amino Acid Sequence↗

PubMed1990

Integration of bacteriophage lambda into the cryptic lambdoid prophages of Escherichia coli.

Bacteriophage lambda missing its chromosomal attachment site will integrate into recA+ Escherichia coli K-12 and C at the sites of cryptic prophages. The specific regions in which these recombination events occur were identified in both lambda and the bacterial chromosomes. A NotI restriction site on the prophage allowed its physical mapping. This allowed us to identify the locations of Rac, Qin, and Qsr' cryptic prophages on the NotI map of E. coli K-12 and, by analogy, to identify the cryptic prophage in E. coli C as Qin. No new cryptic prophages were detected in E. coli K-12.

Bacteriophage lambda↗

PubMed1990

Direct involvement of IS26 in an antibiotic resistance operon.

The plasmid pBWH77, originally found in an isolate of Klebsiella pneumoniae, harbors a new antibiotic resistance operon containing two resistance genes transcribed from an IS26-hybrid promoter, as shown by nucleotide sequencing, mRNA mapping, and the effect of inserting a transcription terminator within the promoter-proximal gene. The nucleotide sequence of this region revealed that the operon (IAB) is made up of three sections that are closely related to previously described genetic elements. The -35 region of the promoter, together with the adjacent sequence, is identical to sequences of the IS26 element. One of the resistance genes, aphA7, which is located next to the hybrid promoter, confers assistance to neomycin and structurally related aminoglycosides. This aphA7 gene is highly homologous to aphA1 of Tn903, with five nucleotide differences. The second gene, blaS2A, encodes an evolved SHV-type beta-lactamase with a pI of 7.6 that confers resistance to the broad-spectrum cephalosporins cefotaxime and ceftizoxime. The deduced amino acid sequence of SHV-2A shows that amino acid 238 is a serine, a residue reported to confer resistance to cefotaxime. We discuss how the operon may have evolved by a combination of insertion sequence-mediated genetic rearrangements and acquisitive evolution. Using phylogenetic parsimony, we show that aphA7 in the IAB operon evolved from an ancestral form similar to aphA1 in Tn903 and that blaS2A evolved from an ancestral form similar to blaS1.

Base Sequence↗

PubMed1990

Membrane association of the Tnp and Inh proteins of IS50R.

Using a radioimmunoassay for the IS50R proteins Tnp and Inh, we found that both proteins were present primarily in the cytoplasm, but 3 to 11% of Tnp and 3 to 5% of Inh were found in association with the inner membrane. The fractions of total Tnp and Inh that became membrane bound were unaffected by the amount of Tnp and Inh synthesized in whole cells, provided that the ratio of total Tnp to total Inh was not changed. In addition, Inh was not found in the membrane fraction in Tnp- IS50R mutants, indicating that Tnp is required for Inh localization.

Bacterial Proteins↗

PubMed1989

Classical plant taxonomic ambiguities extend to the molecular level.

The molecular evolution of cytochrome c from angiosperms is compared to that from vertebrates. On the basis of a cladistic analysis from 26 plant species, compared to that from 27 vertebrate species, we find that although the vertebrate sequences yield reasonably well-defined minimal trees that are congruent with the biological tree, the plant sequences yield multiple minimal trees that are not only highly incongruent with each other, but none of which is congruent with any reasonably biological tree. That is, the plant sequence set is much more homoplastic than that of the animal. However, as judged by the relative rate test, the extent of divergence, and degree of functional constraint, cytochrome c evolution in plants does not appear to differ from that of vertebrates.

Animals↗

PubMed1988

Cointegrate formation by IS50 requires multiple donor molecules.

The insertion sequence, IS50R, promotes cointegrate formation between a lambda::IS50R phage and the chromosome of Escherichia coli strain C. We show that formation of cointegrates mediated by IS50R between the non-replicating phage genome and the bacterial chromosome requires multiple donor molecules and depends on homologous recombination functions. We conclude that the two copies of IS50 present in the cointegrate originate in two different molecules. Thus, the existence of the cointegrate structure cannot be used as evidence for replication of IS50 sequences during IS50 transposition.

Bacteriophage lambda↗

PubMed1988

Functional and structural map of pLST1000: a multiresistance plasmid widely distributed in Enterobacteriaceae.

pLST1000, an 80-kb plasmid found in Enterobacteriaceae in North and South America, harbors the aadB and several other resistance genes. We suggested earlier that, because of its widespread distribution, pLST1000 could act as a carrier plasmid, bringing the aadB gene to new locations. This paper presents the restriction enzyme recognition site and functional map of the plasmid. The resistance genes lie in a discreet region. The aadB and aadA genes form an operon with the aadB gene promoter proximal. This operon is flanked by bla-TEM and bla-OXA2 genes, the former located in a functional Tn3-like transposon. This arrangement is similar to that of relatives of the transposon TN21, where additional resistance genes are precisely inserted in recombinational "hot spot" sequences that flank the aadA gene. We were not able to demonstrate transposition of the aadB gene in Escherichia coli. A sul gene and mer operon lie beyond the bla-OXA2 gene. The transfer genes form a single region, defined by insertions of Tn5-132 that give the Tra- phenotype.

Drug Resistance, Microbial↗

PubMed1988

Temporal control of transposition in Tn5.

IS50R is an insertion sequence associated with the transposon Tn5. IS50R carries the structural genes for two proteins; one (P1) is the Tn5 transposase, and the other (P2) is an inhibitor of transposition. These two proteins are translated from two different transcripts, m1 and m2. When bacteriophage lambda::IS50R DNA was introduced into a bacterial cell, m1 and m2 were initially at relative levels of about 1 to 2. As time progressed the amount of m1 fell, whereas the amount of m2 continued to increase, until after about 3 h the ratio of m1 to m2 was about 1 to 80. The temporal changes in the levels of these transcripts correlated with temporal changes in P1 and P2 levels and Tn5 transposition that have been documented in other studies. We measured the stability of the messages and showed that the differences in the levels of m1 and m2 must reflect real differences in the strengths of their promoters and that the changes in transcription kinetics are mediated by the dam methylation system of the cell and are not determined by IS50R products. Our results show that the 5' end of m2 is about twice as stable as that of m1, which raises the possibility that differential message stability does, in part, influence the ratio of inhibitor to transposase.

Bacterial Proteins↗

PubMed1987

Molecular clocks and evolutionary relationships: possible distortions due to horizontal gene flow.

This paper discusses recent evidence suggesting that genetic information from one species occasionally transfers to another remotely related species. Besides addressing the issue of whether or not the molecular data are consistent with a wide-spread influence of horizontal gene transfer, the paper shows that horizontal gene flow would not necessarily preclude a linear molecular clock or change the rate of molecular evolution (assuming the neutral allele theory). A pervasive influence of horizontal gene transfer is more than just consistent with the data of molecular evolution, it also provides a unique explanation for a number of possibly conflicting phylogenies and contradictory clocks. This phenomenon might explain why some protein clocks are linear while the superoxide dismutase clock is not, how the molecular data on the phylogeny of apes and Australian song birds are not necessarily in conflict with those based on morphology, and, finally, why the mycoplasmas have an accelerated molecular clock.

Animals↗

PubMed1986

Stimulation of precise excision and recombination by conjugal proficient F'plasmids.

Large F plasmids such as F'128 stimulate precise excision of the transposons Tn5 and Tn10 in E. coli K12. This stimulation occurs when the transposons are either on the F'128 plasmid or the bacterial chromosome. Stimulation of precise excision is dependent upon conjugal transfer proficient F'plasmids. Tra- mutations which are defective in conjugal transfer negate this F'128 plasmid stimulation effect. F'128 traS mutations, which are surface exclusion defective and thus permit matings between male cells, thereby increasing conjugal transfer, increase the F plasmid stimulation effect. When the F' plasmid is present in a cell with the small plasmid, pRS31, carrying the traS to traZ region of F, stimulation of precise excision is no longer observed. This complementation-like activity by pRS31 is abolished by a Tn5 insertion in the traS gene. Data are presented supporting the notion that F' plasmid stimulation of precise excision occurs in the recipient during conjugal transfer. F'128 traS also stimulates recA-dependent recombination between DNA sequences on the small, nontransferrable plasmid pRDK41, DNA sequences that are unrelated to those of the F plasmid. The F'plasmid stimulation of precise excision of Tn5 is not seen with F+ but only with certain F's with large insertions of chromosomal DNA.

Conjugation, Genetic↗

PubMed1986

Genetic and physical characterization of IncM plasmid pBWH1 and its variance among natural isolates.

We present a genetic and physical characterization of the IncM plasmid pBWH1. A physical map was constructed for the enzymes EcoRI, BamHI, SalI, BglII, HindIII, MstII, and XhoI. A series of deletions and a series of subclones of pBWH1 were constructed and used to determine the locations on this map of the transfer region; the replication region; and the genes determining resistance to beta-lactams, chloramphenicol, the sulfonamides, and gentamicin. We compared 51 different isolates, including isolates which had lost individual antibiotic resistances or the transfer phenotype, and showed that variations occurred in all regions of the plasmid genome. Frequently, correlations could be made between phenotypic variation and variation of the EcoRI fragments which contained the gene determining that phenotype.

Chromosome Mapping↗

PubMed1985

Intercontinental spread of a new antibiotic resistance gene on an epidemic plasmid.

Bacteria of different genera isolated at nine medical centers in different parts of the United States and at one center in Venezuela during the first decade of gentamicin usage carried the gentamicin resistance gene 2"-aminoglycoside nucleotidyltransferase on the same transferable plasmid. Such widespread dissemination of a newly observed resistance gene on one plasmid suggests that a new resistance gene may emerge once on a single plasmid, which then carries it to other centers and other plasmids. The resistance gene might, therefore, be contained if detected early.

Bacteria↗