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PubMed2026

Genomic characterization of novel human-associated CTX-M-15-producing Serratia nevei ST625 lineage infecting a vulnerable loggerhead sea turtle.

BACKGROUND: Serratia nevei is a newly classified and opportunistic bacterial species belonging to the Serratia marcescens complex (SMC). Genomic data from this species is highly relevant for public health and epidemiological tracking. OBJECTIVE: To report the first identification and genomic characterization of extended-spectrum β-lactamase (CTX-M-15)-producing S. nevei sequence type (ST) ST625 lineage infecting a vulnerable loggerhead sea turtle. METHODS: Strain BP02 was recovered from the coelomic cavity of a loggerhead sea turtle (Caretta caretta) admitted to a rehabilitation center in southeastern Brazil. MALDI-TOF MS was initially used for species identification and was further confirmed by whole-genome sequencing on the Illumina HiSeq platform, followed by ANI, dDDH, multilocus sequence typing, resistome, plasmidome, virulome, and SNP-based phylogenomic analyses. RESULTS: Strain BP02 exhibited a multidrug-resistant profile, including resistance to third- and fourth-generation cephalosporins. Genomic analyses identified BP02 as S. nevei ST625 carrying blaCTX-M-15 within the ISEcp1-blaCTX-M-15-wbuC-ΔTn2 genetic environment, in addition to multiple AMR determinants and the IncC plasmid replicon. Phylogenomic analysis demonstrated close relatedness between BP02 and human clinical ST625 strains, previously reported in São Paulo, Brazil, including a urine-derived strain isolated in 2019, differing by only 27 SNPs. Notably, all publicly available ST625 genomes were associated with human clinical sources and displayed multidrug resistance genotypes. CONCLUSION: This study expands the current knowledge regarding the ecology and genomic features of S. nevei, demonstrating the emergence of a human multidrug-resistant clone in marine wildlife. Our findings reinforce the importance of monitoring clinically relevant SMC members across distinct ecological niches within a One Health perspective.

ESBL↗

PubMed1988

Localization of the pro-sequence within the total deduced primary structure of human beta-hexosaminidase B.

The beta subunit of beta-hexosaminidase (beta-N-acetylhexosaminidase, EC 3.2.1.52) is synthesized in the rough endoplasmic reticulum as a prepropolypeptide. After the loss of the signal peptide and formation of an enzymatically active dimer, the pro-enzyme is either secreted from the cell or transported into the lysosome for processing to its mature form. In order to characterize the early posttranslational events we have purified nearly 1 mg of pro-hexosaminidase B from the NH4Cl containing medium of fibroblasts derived from a patient with the infantile form of Tay-Sachs disease. The partial N-terminal sequence was mapped to a position 42 residues C-terminal to the first in-frame ATG (Met residue) and 79 residues N-terminal to the known mature N-terminus. This position corresponds to that predicted for the cleavage of a 17 amino acid signal peptide generated through the use of the third rather than the first in-frame ATG as the initiation site for protein synthesis.

Amino Acid Sequence↗

PubMed1995

Multiple substitutions at position 104 of beta-lactamase TEM-1: assessing the role of this residue in substrate specificity.

Residue 104 is frequently mutated from a glutamic acid to a lysine in the extended-spectrum TEM beta-lactamases responsible for the resistance to third-generation cephalosporins in clinical Gram negative strains. Among class A beta-lactamases, it is the most variable residue within a highly conserved loop which delineates one side of the active site of the enzymes. To investigate the role of this residue in the extended-spectrum phenotype, it has been replaced by serine, threonine, lysine, arginine, tyrosine and proline. All these substitutions yield active enzymes, with no drastic changes in kinetic properties compared with the wild-type enzyme, except with cefaclor, but an overall improved affinity for second- and third-generation cephalosporins. Only mutant E104K exhibits a significant ability to hydrolyse cefotaxime. Molecular modelling shows that the substitutions have generally no impact on the conformation of the 101-111 loop as the side chains of residues at position 104 are all turned towards the solvent. Unexpectedly, the E104P mutant turns out to be the most efficient enzyme. All our results argue in favour of an indirect role for this residue 104 in the substrate specificity of the class A beta-lactamases. This residue contributes to the precise positioning of residues 130-132 which are involved in substrate binding and catalysis. Changing residue 104 could also modify slightly the local electrostatic potential in this part of the active site. The limited kinetic impact of the mutations at this position have to be analysed in the context of the microbiological problem of resistance to third-generation cephalosporins. Although mutation E104K improves the ability of the enzyme to hydrolyse these compounds, it is not sufficient to confer true resistance, and is always found in clinical isolates associated with at least one mutation at another part of the active site. It is the combined effect of the two mutations that synergistically enhances the hydrolytic capability of the enzyme towards third-generation cephalosporins.

Amino Acid Sequence↗

PubMed1994

Multiplex PCR assay specific for the multidrug-resistant strain W of Mycobacterium tuberculosis.

In 1991, a multidrug-resistant strain of Mycobacterium tuberculosis was isolated from eight people with tuberculosis at a state correctional facility in New York. This strain, which is designated strain W (IS6110 restriction fragment length polymorphism type 212072), was resistant to isoniazid, rifampin, ethambutol, streptomycin, kanamycin, ethionamide, and rifabutin. Since that outbreak, the W strain has been associated with outbreaks in five hospitals in the New York City area and is a continuing public health problem in the area. To be able to identify this strain rapidly, we developed a multiplex PCR assay which targets a direct repeat of IS6110 with a 556-bp intervening sequence (NTF-1). The amplification generates two amplicons from strain W, which indicate the presence and orientation of the NTF-1 sequence between the direct repeat of IS6110, and a third amplicon, which serves as an internal PCR control. The assay was evaluated with 193 isolates of M. tuberculosis, and all 48 strain W isolates among those 193 isolates were correctly identified.

Antitubercular Agents↗

PubMed1993

A major difference between the divergence patterns within the lines-1 families in mice and voles.

L1 retroposons are represented in mice by subfamilies of interspersed sequences of varied abundance. Previous analyses have indicated that subfamilies are generated by duplicative transposition of a small number of members of the L1 family, the progeny of which then become a major component of the murine L1 population, and are not due to any active processes generating homology within preexisting groups of elements in a particular species. In mice, more than a third of the L1 elements belong to a clade that became active approximately 5 Mya and whose elements are > or = 95% identical. We have collected sequence information from 13 L1 elements isolated from two species of voles (Rodentia: Microtinae: Microtus and Arvicola) and have found that divergence within the vole L1 population is quite different from that in mice, in that there is no abundant subfamily of homologous elements. Individual L1 elements from voles are very divergent from one another and belong to a clade that began a period of elevated duplicative transposition approximately 13 Mya. Sequence analyses of portions of these divergent L1 elements (approximately 250 bp each) gave no evidence for concerted evolution having acted on the vole L1 elements since the split of the two vole lineages approximately 3.5 Mya; that is, the observed interspecific divergence (6.7%-24.7%) is not larger than the intraspecific divergence (7.9%-27.2%), and phylogenetic analyses showed no clustering into Arvicola and Microtus clades.

Animals↗

PubMed1993

Mechanisms of oncogenesis in patients with familial retinoblastoma.

In an analysis of mutations in the RB1 gene in three patients, selected at random, who had a positive family history of tumours, we identified mutations, in constitutional cells, involving exons 3, 13 and 17 of the RB1 gene. We used SSCP and PCR sequencing to screen affected individuals and other members of their families. In two cases the mutations were 2 bp and 1 bp deletions identified in exons 3 and 17 respectively. The third mutation was a 1 bp insertion in exon 13. All three mutations lead to the generation of downstream premature stop codons as a result of frameshift changes, although the mutation in exon 3 possibly affects the splicing mechanism. The sites within the RB1 gene where these mutations occur contain interspersed repetitive DNA sequences, direct and inverted repeat sequences and/or dyad symmetrical elements suggesting that these areas promote the appropriate local sequence environment for the generation of deletions and insertions in the RB1 gene.

Amino Acid Sequence↗

PubMed1992

Recognition of a tetranucleotide loop of signal recognition particle RNA by protein SRP19.

The interaction of protein SRP19 with the RNA component of human signal recognition particle (SRP) was studied by site-directed mutagenesis of the SRP RNA. The effects of nucleotide changes in the tetranucleotide loop (tetraloop) of helix 6 showed that SRP19 recognizes a tetraloop in a sequence-specific manner. Adenosine 149 at the third position of the tetraloop was essential for binding. In contrast, changes of the base at the second position had no effect. Mutations that disrupt or compensate individual SRP RNA helices were generated to investigate the importance of base pairing and to identify other binding sites. Considerable base pairing was essential in helix 6. Another SRP19-binding site was located in the distal part of helix 8. The primary sequences of the tetraloop-binding protein SR19 and of bacterial ribosomal protein S15 are shown to be similar.

Amino Acid Sequence↗

PubMed1991

Use of high coverage reference libraries of Drosophila melanogaster for relational data analysis. A step towards mapping and sequencing of the genome.

Three differently made, primary Drosophila cosmid libraries of 16-fold genome coverage have been generated. Also, a jumping library has been created by a new method that takes advantage of methylation differences between genomic DNA and vector. Thirdly, two cDNA libraries have been picked. All these libraries have been arrayed on high-density in situ filters, each containing 9216 clones. As a reference system, such filters are distributed and identified clones are provided. Single-copy probes have identified on average 1.4 cosmids per genome equivalent. Together with cytogenetically mapped yeast artificial chromosomes, the libraries are also being used for physically mapping the genome, mainly by oligonucleotide fingerprinting and pool hybridizations. cDNA clones are further examined by a partial sequencing analysis by oligomer hybridization.

Animals↗

PubMed1994

A molecular phylogeny of the gopher tortoises, with comments on familial relationships within the Testudinoidea.

Sequences from the mitochondrial cytochrome b gene were obtained to examine molecular phylogenetic relationships among the North American gopher tortoises. Data from 352 aligned positions generated a single most-parsimonious tree for each of three analytical approaches: (1) equal weighting, all substitutions; (2) equal weighting, third position changes limited to transversions; and (3) transversions weighted 10 times transitions. Identical topologies for the resulting trees depict the gopher tortoises as a monophyletic group comprising two well-defined clades. Observed sequence divergence (7.0%) between the agassizii (Gopherus agassizii; G. berlandieri) and the polyphemus (G. flavomarginatus; G. polyphemus) clades suggests an Early Miocene separation of these lineages. The cytochrome b phylogeny complements some previous systematic interpretations, including formal taxonomic recognition of the two distinct groups, but is at odds with the most recent morphological analysis. Additional sequence comparisons of selected testudinoid (batagurid, emydid, and testudinid) taxa yielded a phylogeny consistent with a morphologically based hypothesis demonstrating close phylogenetic affinities between the Testudinidae and the "Bataguridae."

Animals↗

PubMed1993

Cloning of the rat aortic smooth muscle Na+/Ca2+ exchanger and tissue-specific expression of isoforms.

The amino acid sequences of two isoforms of the rat aortic smooth muscle Na+/Ca2+ exchanger have been deduced by cloning and sequencing the cDNAs. These isoforms are identical in nucleotide sequence except that one has a 23-amino acid insertion at amino acid position 570. They are highly homologous to the canine cardiac exchanger except for the NH2-terminal portion and part of the large central hydrophilic domain (amino acid residues 570-631). They are 902 and 925 (with the insertion) amino acid long with calculated molecular masses of 100,676 and 103,200 (with the insertion), respectively, if the NH2-terminal 32-amino acid residues are eliminated as a cleaved signal sequence. Amplification of the variable region (amino acids 570-631) of the exchanger by means of the reverse transcriptase-polymerase chain reaction and DNA sequencing revealed that many isoforms of the exchanger are expressed in different rat tissues. The two clones isolated in this study are the predominant isoforms expressed in aorta, stomach, liver, and kidney. In cardiac and skeletal muscles, another isoform is dominant, which is equivalent to the canine cardiac exchanger. In brain, a third type is predominantly expressed. Alignment of the nucleotide sequences of these isoforms and Southern blot analysis of rat genomic DNA suggested that each isoform is generated through alternative splicing of the primary transcript.

Alternative Splicing↗

PubMedDate not supplied

The mutation causing the common apolipoprotein A-IV polymorphism is a glutamine to histidine substitution of amino acid 360.

Apolipoprotein (apo) A-IV is a protein involved in the metabolism of chylomicrons and high density lipoproteins. This protein displays genetic polymorphism due to two main codominant alleles, A-IV1 and A-IV2. We have identified the mutation that leads to this polymorphism. It is caused by a single-base substitution of guanine for thymine in the third base of codon 360. This substitution leads to a glutamine to histidine change. Direct sequencing of amplified DNA from eight subjects in a three-generation pedigree has demonstrated that the guanine to thymine substitution can explain the apo A-IV polymorphism. In 32 unrelated individuals, a correspondence between apo A-IV phenotype determined by isoelectric focusing and genotype determined with Fnu4HI digestion of amplified DNA could be demonstrated. The enzyme lecithin:cholesteryl acyltransferase (LCAT) is activated by apo A-IV. Under our in vitro conditions, the isoprotein apo A-IV 1-1 is a better LCAT activator than is the isoprotein apo A-IV 2-2. A knowledge of the molecular mechanism underlying the apo A-IV polymorphism will help to elucidate the mechanisms involved in LCAT activation.

Amino Acid Sequence↗

PubMed1990

Detection of minimal residual disease in acute lymphoblastic leukemia using immunoglobulin hypervariable region specific oligonucleotide probes.

To develop a sensitive and specific assay for minimal residual disease in acute lymphoblastic leukemia (ALL), we exploited the enormous diversity of genomic sequences created by immune receptor gene rearrangements. To isolate clone-specific sequences, we first synthesized oligonucleotides that match conserved variable (VH) and joining (JH) sequences flanking the third hypervariable region (HVR3) in the rearranged immunoglobulin heavy chain (IgH) locus. In polymerase chain reactions (PCR), these primers were then used to amplify the intervening HVR3 segments from leukemic DNA samples. Of 12 B-lineage ALLs studied, ten generated one or more fragments of the size expected for HVR3 gene segments. Thus, this single pair of amplimers was sufficient to isolate HVR3 sequences from a majority of acute lymphoblastic leukemias. To verify that the amplified fragments originated from HVR3 alleles and to assess their diversity, we sequenced 7 PCR products derived from 6 leukemias. In addition to elements of recognized D segments, each of the 7 fragments contained novel VH-D and D-JH junctional sequences, including N nucleotides, not known to be present in the germline. Each sequence was unique, and allele-specific oligonucleotide probes hybridized only to HVR3 segments from which the probes were derived. Therefore, as anticipated, these HVR3 segments appeared to possess the diversity required to serve as clonal markers for leukemic populations. To demonstrate that these amplified HVR3 alleles could serve as the basis for a sensitive and specific assay to detect rare leukemic cells, we analyzed in detail one pre-B leukemia that had rearranged 2 IgH alleles. The HVR3 sequences were shown to be linked to rearranged JH-containing restriction fragments in digests of genomic DNA, establishing their origin in the leukemic cells. We synthesized oligonucleotides corresponding to the unique junctional sequences in the HVR3 segments. Using these novel amplimers in an allele-specific amplification and hybridization procedure, we showed that this assay can detect 10 leukemic cells in a background of 10(6) normal blood mononuclear cells. In contrast, the leukemic HVR3 sequences were not detected in extracts of normal or unrelated remission leukemic leukocytes. We conclude that the assay for specific IgH HVR3 sequences is a realistic strategy for detection of minimal residual disease in B-lineage ALL.

Alleles↗

PubMed1990

Molecular structure of microtubule-associated protein 2b and 2c from rat brain.

Full length cDNA clones encoding microtubule-associated proteins (MAP) 2b and 2c from rat brain have been isolated and sequenced. The cDNA fragments spanning the coding regions for both MAP2b and MAP2c were assembled and expressed in Escherichia coli. The mobility of these bacterial expressed proteins in sodium dodecyl sulfate gels is identical to that of MAP2b and MAP2c from rat brain. The protein sequence of rat MAP2b has been compared to the full length sequence from mouse and the partial sequence from human high molecular weight MAP2. This comparison has revealed that MAP2b is composed of several highly conserved domains flanked by domains with extensive sequence divergence. Two of the conserved domains, found either at the NH2 or COOH terminus, overlap with the binding domain for the regulatory subunit of the cAMP-dependent protein kinase II and the microtubule-binding domain, respectively. A third homologous domain of unknown function lies in a central region of MAP2b. Secondary structure prediction suggests that the portion of MAP2b which extends from the microtubule surface is composed of an extensive number of alpha-helices separated by small turns which may account for the extended yet flexible structure of MAP2. Interestingly, the 4000-base pair deletion from the middle of MAP2b which generates MAP2c not only removes these helices, but also this third highly conserved MAP2b domain.

Amino Acid Sequence↗

PubMed1992

Differential splicing in the extracellular region of fibroblast growth factor receptor 1 generates receptor variants with different ligand-binding specificities.

We have cloned a genomic region of the murine fibroblast growth factor (FGF) receptor 1 (FGFR1) gene that includes three alternative exons for the third immunoglobulinlike domain in the extracellular region of the receptor. The mRNA of one of these splice variants encodes a secreted receptor that lacks transmembrane and cytoplasmic sequences as well as a portion of the third immunoglobulinlike domain. Highest levels of mRNA encoding this variant were found in brain, skeletal muscle, and skin. We expressed this form of FGFR1 in CHO cells and showed that the recombinant secreted protein binds acidic FGF. We also discovered a novel alternative exon in the third immunoglobulinlike domain that encodes part of a transmembrane FGFR1 mRNA. This exon is highly homologous to the corresponding region of the keratinocyte growth factor receptor. Transcripts including this exon were present at highest levels in the skin. We cloned an FGFR1 cDNA which includes this exon and expressed this receptor variant in L6 rat skeletal muscle myoblasts. The new receptor variant had a 50-fold-lower affinity for basic FGF than does the published FGFR1 variant, whereas both forms of receptor bound acidic FGF with high affinity. These results show that the third immunoglobulinlike domain plays an important role in determining the binding specificities for different FGFs. Our data provide the first evidence that differential splicing in the extracellular region of a receptor gene generates receptor variants with different ligand-binding specificities.

Amino Acid Sequence↗

PubMed1991

P300 generation by novel somatosensory stimuli.

Event-related potentials (ERPs) to task-relevant target and task-irrelevant novel stimuli were recorded in a somatosensory discrimination task. Subjects pressed a button to mechanical taps of the fifth finger (targets, P = 0.12), randomly interposed in sequences of taps to the second finger (standards, P = 0.76). Two types of infrequent novel stimuli were delivered; one was a mechanical tap to the third or fourth finger (tactile novels, P = 0.06), another was an electric shock at the wrist (shock novels, P = 0.06). Correctly detected targets generated a parietal maximal P300 (P3b, latency 335 msec). Shock novels generated a central maximal P300 with a shorter peak latency (298 msec) than the P3b. Tactile novels generated a P300 with a scalp distribution comparable to the shock novels. Unlike the P3b, P300 amplitude to both the shock and tactile novel stimuli habituated by 20-30% across the first several stimulus presentations. These results indicate that, similar to the auditory and visual modality, task-irrelevant novel somatosensory stimuli generate a novelty P300 ERP. Differences in scalp distribution, latency and habituation characteristics suggest that the novelty P300 may have contributions from intracranial generators independent from target P300 sources.

Adolescent↗

PubMed1981

Identification of D segments of immunoglobulin heavy-chain genes and their rearrangement in T lymphocytes.

The finding that the diversity (D) and joining (JH) but not the variable (VH) DNA segments of mouse immunoglobulin heavy-chain genes are joined in the DNA of some cloned cytolytic T cells, led to identification and sequencing of three different D DNA segments. Two segments identified on the embryo DNA carry on both the 5' and 3' sides two sets of characteristic sequences separated by a 12-base pair spacer, which have been implicated as recognition signals for a recombinase. The third segment, identified in a form joined with a JHDNA segment in a T cell, carries the recognition signal on the 5' side. These results support the 12/23-base pair model for somatic generation of immunoglobulin V genes, and rule out the possibility that the cytolytic T cells use assembled VH, D and JH sequences to encode their antigen receptors.

Animals↗

PubMed1994

Alternative splicing generates two distinct transcripts for the Drosophila melanogaster fibroblast growth factor receptor homolog.

We screened Drosophila melanogaster genomic and cDNA libraries by low-stringency hybridization with a probe representing the protein tyrosine kinase (TyK) domain encoded by a human alpha-platelet-derived growth factor receptor-encoding cDNA. The complete sequences of the open reading frames and 3'-untranslated regions (UTR) of some cross-hybridizing clones were identical to the recently published sequence of DFR1, encoding the novel D. melanogaster fibroblast growth factor receptor homology. However, two species of DFR1 cDNAs were isolated that differed with respect to their 5'-UTR. Analysis of the genomic organization revealed that DFR1 is composed of three exons. The entire coding region is contained within the third exon. S1 mapping and RNase-protection assays demonstrated that two distinct DFR1 transcripts possessing either the first or the second exon in combination with the third exon are generated by alternative splicing. This suggests that the transcriptional, as well as posttranscriptional, regulation of fibroblast growth factor receptor (FGFR)-encoding genes during D. melanogaster development is likely to be complex.

Alternative Splicing↗

PubMed1988

Ancient origin of lactalbumin from lysozyme: analysis of DNA and amino acid sequences.

Parsimony trees relating DNA sequences coding for lysozymes c and alpha-lactalbumins suggest that the gene duplication that allowed lactalbumin to evolve from lysozyme preceded the divergence of mammals and birds. Comparisons of the amino acid sequences of additional lysozymes and lactalbumins are consistent with this view. When all base positions are considered, the probability that the duplication leading to the lactalbumin gene occurred after the start of mammalian evolution is estimated to be 0.05-0.10. Elimination of the phylogenetic noise generated by fast evolution and compositional bias at third positions of codons reduced this probability to 0.002-0.03. Thus the gene duplication may have long preceded the acquisition of lactalbumin function.

Animals↗