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PubMed2025

Comparative genomics of carbapenem-resistant Acinetobacter baumannii isolated from pediatric patients in a tertiary care hospital.

Acinetobacter baumannii is a short gram-negative bacillus, notable for its intrinsic multidrug resistance and genomic plasticity, which facilitates the acquisition of additional resistance genes via mobile genetic elements. Due to its increasing carbapenem resistance, the World Health Organization has classified it as a critical priority pathogen. This study performed a comparative genomic analysis of 20 carbapenem-resistant A. baumannii clinical strains isolated from the Hospital Infantil de México Federico Gómez (CRAB-HIMFG), alongside 11 genomes from other Mexican strains. The pangenome was determined to be open, and core genome single-nucleotide polymorphism-based analysis grouped the CRAB-HIMFG strains within CC758/IC5 and CC92/IC2. A novel sequence type (ST) in the MLST-Pasteur scheme was identified, related to STPas156, and in the MLST-Oxford scheme, associated with STOxf758 and STOxf1054. Virulence and resistance genes comprised 0.61% to 2.23% of the pangenome. Oxacillinase genes and efflux pumps primarily mediated carbapenem resistance, while virulence genes included those encoding biofilm and type IV pili. Capsule typing revealed a correlation with established international clones, IC2 and IC5. Plasmids exhibited high diversity, harboring maintenance modules and toxin-antitoxin systems, with the dissemination of resistance genes linked to insertion sequences. Biofilm formation and twitching motility were not always expressed, as they depend on additional environmental factors. Our study shows that comparative genomics is an essential tool to analyze clinically and epidemiologically significant genomes, providing critical insights into gene distribution, genomic architecture, and horizontal gene transfer mechanisms in microbial populations.IMPORTANCEIn recent years, a reported increase in the mortality rate associated with infections caused by A. baumannii, along with a rise in carbapenem resistance, poses a serious clinical challenge. The WHO considered this microorganism critical for research into alternative therapies and epidemiological surveillance. Despite advances in bioinformatics, genomic studies have yet to fully elucidate the structural rearrangements and secretion systems of A. baumannii. This knowledge gap hinders our understanding of its remarkable genomic plasticity and its ability to acquire and spread resistance and virulence genes through horizontal gene transfer.

Acinetobacter baumannii↗
PubMed2001

Selective 'stencil'-aided pre-PCR cleavage of wild-type sequences as a novel approach to detection of mutant K-RAS.

The enriched PCR widely used for detection of mutant K-RAS in either tumor tissues or circulating DNA was modified so that abundant wild-type K-RAS alleles are cleaved prior to PCR. We took advantage of an AluI recognition site located immediately upstream of the K-RAS codon 12. The site was reconstituted upon DNA denaturation followed by annealing with a 'stencil', a 16-bp synthetic oligonucleotide complementary to the wild-type sequence. As opposed to normal K-RAS, the mutant allele forms, upon annealing with the stencil, a mismatch at the codon 12 which lies within the AluI enzyme binding site and partially inhibits its activity. The mismatch also lowers the melting temperature of the stencil-mutant K-RAS double helix as compared to stencil-wild-type duplex, so that only the latter is double stranded and selectively digested by AluI at elevated temperatures. The proposed method of stencil-aided mutation analysis (SAMA) based on selective pre-PCR elimination of wild-type sequences can be highly advantageous for detection of mutant K-RAS due to: (i) an enhanced sensitivity because of reduced competition with a great excess of normal K-RAS, and (ii) a decrease in a number of false-positive results from Taq polymerase errors. Application of SAMA for generalized detection of DNA mutations is discussed.

Base Sequence↗
PubMed2003

Purification, characterization, and sequencing of a novel type of antimicrobial peptides, Fa-AMP1 and Fa-AMP2, from seeds of buckwheat (Fagopyrum esculentum Moench.).

Novel antimicrobial peptides (AMP), designated Fa-AMP1 and Fa-AMP2, were purified from the seeds of buckwheat (Fagopyrum esculentum Moench.) by gel filtration on Sephadex G75, ion-exchange HPLC on SP COSMOGEL, and reverse-phase HPLC. They were basic peptides having isoelectric points of over 10. Fa-AMP1 and Fa-AMP2 had molecular masses of 3,879 Da and 3,906 Da on MALDI-TOF MS analysis, and their extinction coefficients in 1% aqueous solutions at 280 nm were 42.8 and 38.9, respectively. Half of all amino acid residues of Fa-AMP1 and Fa-AMP2 were cysteine and glycine, and they had continuous sequences of cysteine and glycine. The concentrations of peptides required for 50% inhibition (IC50) of the growth of plant pathogenic fungi, and Gram-positive and -negative bacteria were 11 to 36 microg/ml. The structural and antimicrobial characteristics of Fa-AMPs indicated that they are a novel type of antimicrobial peptides belonging to a plant defensin family.

Actinomycetales↗
PubMed2001

Structural analyses of the deduced amino acid sequences of a novel type heme-copper terminal oxidase, cytochrome aco3, from alkalophilic Bacillus YN-2000.

Cytochrome aco3 from a facultatively alkalophilic bacterium, Bacillus YN-2000, was found to be alkaline- and heat-tolerant. To better understand the structural features of Bacillus YN-2000 cytochrome aco3, the gene encoding this enzyme was cloned and sequenced. Nucleotide sequence analyses of the region neighboring the acoI (subunit I) gene revealed that the acoII (subunit II) and acoIII (subunit III) genes were concomitantly clustered upstream and downstream of the acoI gene, respectively, forming an operon with transcriptional polarity. The deduced amino acid sequence of subunit I was highly similar to that of cytochrome caa3 from thermophilic bacterium Bacillus PS3 in which the heme a3 could be replaced with heme o. Furthermore, a marked paucity of basic amino acid residues was found in the cytochrome c-binding subunit II, which might be a result of the adaptation to a highly alkaline external milieu.

Amino Acid Sequence↗
PubMed1992

A novel sequence in the type IV collagen promoter binds nuclear proteins from Engelbreth-Holm-Swarm tumor.

The production of extracellular matrix proteins is an important element of tumor formation, and alterations in matrix protein metabolism may be critical to the process of tumor metastasis. Abundant expression of type IV collagen, the major structural protein of the basement membrane, is characteristic of the Engelbreth-Holm-Swarm (EHS) mouse sarcoma. In the present study, we evaluated mechanisms of transcriptional regulation of type IV collagen genes by analysing nuclear factors that bind to the promoter region. Gel mobility-shift assays indicated that specific proteins from EHS tumor bound the promoter and generated several unique shift patterns. The specific sequences to which these proteins bound were determined using DNAase I protection assays. DNA-binding proteins protected two regions from DNAase I digestion. The first region was similar to a GC box, the binding site for the transcription factor Sp1. The other footprint was a 30-bp region that contained the novel sequence motif, 'CCCTCCC' present in several other extracellular matrix promoters. Nuclear extracts isolated from tissues that variably express type IV collagen bound to this protected sequence with distinctly different shift patterns. Furthermore, in highly expressing tissues, unlabeled oligonucleotides containing the 'CCCTCCC' motif effectively inhibited nuclear protein binding with the entire promoter. Thus, it is likely that a novel protein or protein complex binds to these sequences. Furthermore, these sequences appear to be unique to the genes that encode basement membrane proteins, suggesting a specific role in their regulation.

Animals↗
PubMed1993

Characterization of the patterns of polymorphism in a "cryptic repeat" reveals a novel type of hypervariable sequence.

Alternating purine and pyrimidine repeats (RY(i)) are an abundant source of polymorphism. The subset with long tandem repeats of GT or AC (GT(i)) have been studied extensively, but cryptic RY(i) (i.e., no single tandem repeat predominates) have received little attention. The factor IX gene has a polymorphic cryptic RY(i) of 142-216 bp. Previously, there were four known polymorphic alleles, of the form AB, A2B, A2B2, and A3B2, where A = (GT)(AC)3(AT)3(GT)(AT)4 and B = A with an additional 3' AT dinucleotide. To further characterize this locus, we examined more than 1,700 additional human chromosomes and determined the sequences of the homologous sites in orangutans and chimpanzees. The novel alleles found in humans expand the repertoire of A/B alleles to A0-4B1 and A1-3B2. The AnB2 series are abundant in Caucasians but are absent in blacks and Asians. Conversely, the A0B1 allele is common in blacks but is not found in more than 1,700 Caucasian chromosomes. The data are compatible with a model in which recombination is more frequent than polymerase slippage at this locus. In orangutans, the RY(i) is present, but the sequence is markedly different. An A/B-type of pattern was discerned in which B differs from A by an additional six (AT) dinucleotides at the 3' end. In chimpanzees, the size of the RY(i) locus was greatly expanded, and the sequence showed a novel pattern of hypervariability in which there are many tandem repeats of the form (GT)n(AC)o(AT)p(GT)q(AT)s, where n, o, p, q, and s are different integers. The sequences of the factor IX intron 1 cryptic RY(i) in three primates provide perspective on the range of possible patterns of polymorphism. Analysis of the patterns suggests how the RY(i) can be conserved during evolution, while the precise sequence varies.

Alleles↗
PubMed1981

Characterization and distribution of nucleic acid sequences of a novel type C retrovirus isolated from neoplastic human T lymphocytes.

A type C retrovirus (designated HTLV) recently isolated from a cell line derived from a lymph node and later from peripheral blood of a person with cutaneous T-cell lymphoma (mycosis fungoides) was characterized by nucleic acid hybridization experiments. HTLV [3H]cDNA hybridized 90% to its own 70S RNA with kinetics consistent with the genetic complexity of other retroviruses, but it did not hybridize substantially to RNA or proviral DNA from any animal retroviruses (types B, C, and D), including those from nonhuman primates. Conversely, [3H]cDNA from other retroviruses did not hybridize to RNA or DNA of the human T-cell line producing HTLV. HTLV proviral sequences were present (two to three copies per haploid genome) in DNA of these cells, and homologous sequences were present in the cell cytoplasmic RNA (0.3% viral sequences by weight). HTLV-related nucleic acid sequences were not found in DNA from various other human tissues. The results indicate that HTLV is a new class of type C virus that is not an endogenous (genetically transmitted) retrovirus in man.

Base Sequence↗
PubMed2005

Identification of a novel class of target genes and a novel type of binding sequence of heat shock transcription factor in Saccharomyces cerevisiae.

In response to hyperthermia, heat shock transcription factor (HSF) activates transcription of a set of genes encoding heat shock proteins (HSPs). The promoter regions of HSP genes contain the HSF binding sequence called the heat shock element (HSE), which consists of contiguous inverted repeats of the sequence 5'-nGAAn-3' (where n is any nucleotide). We have constructed an hsf1 mutant of Saccharomyces cerevisiae and analyzed genome-wide changes in heat shock response in the mutant cells. The results have revealed that Hsf1 is necessary for heat-induced transcription of not only HSP but also genes encoding proteins involved in diverse cellular processes such as protein degradation, detoxification, energy generation, carbohydrate metabolism, and maintenance of cell wall integrity. Approximately half of the Hsf1-regulated genes lacked the typical HSE in their promoter regions. Instead, several of these genes have a novel Hsf1 binding sequence that contains three direct repeats of nTTCn (or nGAAn) interrupted by 5 bp. The number and spacing of the repeating units are critical determinants for heat-induced transcription as well as for recognition by Hsf1. In the yeast genome, the presence of the sequence is enriched in Hsf1-regulated genes, suggesting that it is generally used as an HSE in the Hsf1 regulon.

Base Sequence↗
PubMed1990

N-terminal amino acid sequence of the novel type IIIb trimethoprim-resistant plasmid-encoded dihydrofolate reductase from Shigella sonnei.

The type IIIb dihydrofolate reductase, a novel plasmid-encoded enzyme recently identified in Shigella sonnei, has been shown to have some similar biochemical properties to the type IIIa dihydrofolate reductase which was first identified in New Zealand in 1979. However, the type IIIb enzyme has a Ki for trimethoprim of 0.4 microM, and a pI of 5.35 (as compared to 19 nM and 6.1 for the type IIIa); both these results suggest that it is a different enzyme from the prototype type IIIa. The type IIIb dihydrofolate reductase was purified by methotrexate agarose affinity chromatography, yielding a pure protein as determined by HPLC. Automatic amino acid analysis of the purified enzyme showed it to be distinct from all other known plasmid-encoded dihydrofolate reductases and quite different from the type IIIa enzyme. The purified enzyme was examined by SDS-PAGE, which revealed that the type IIIb dihydrofolate reductase was a monomeric protein of Mr 17,200.

Amino Acid Sequence↗
PubMed2003

Engineering of restriction endonucleases: using methylation activity of the bifunctional endonuclease Eco57I to select the mutant with a novel sequence specificity.

Type II restriction endonucleases (REs) are widely used tools in molecular biology, biotechnology and diagnostics. Efforts to generate new specificities by structure-guided design and random mutagenesis have been unsuccessful so far. We have developed a new procedure called the methylation activity-based selection (MABS) for generating REs with a new specificity. MABS uses a unique property of bifunctional type II REs to methylate DNA targets they recognize. The procedure includes three steps: (1) conversion of a bifunctional RE into a monofunctional DNA-modifying enzyme by cleavage center disruption; (2) mutagenesis and selection of mutants with altered DNA modification specificity based on their ability to protect predetermined DNA targets; (3) reconstitution of the cleavage center's wild-type structure. The efficiency of the MABS technique was demonstrated by altering the sequence specificity of the bifunctional RE Eco57I from 5'-CTGAAG to 5'-CTGRAG, and thus generating the mutant restriction endonuclease (and DNA methyltransferase) of a specificity not known before. This study provides evidence that MABS is a promising technique for generation of REs with new specificities.

Amino Acid Motifs↗
PubMed1990

Scripts or scraps: reconsidering the development of sequential understanding.

A growing literature attests to temporally ordered recall of events by children under 2 years of age. Other data suggest a developmental sequence wherein the ability to reproduce unfamiliar and/or arbitrarily ordered events, and familiar events in other than canonical order develops well after the first ordered productions of events. Early ordering is thus argued to be dependent upon familiarity, rather than upon general temporal principles. This suggestion was investigated by using elicited imitation to assess 21-month-olds' recall of familiar-canonical, familiar-reversed, novel-causal, and novel-arbitrary event sequences. Subjects reproduced canonical and both types of novel sequences in modeled order. On reversed sequences they vacillated between reproducing the events as modeled and "correcting" them to canonical order. The results suggest that temporal organization is not imposed upon an existing unordered event representation, but rather, is an integral aspect of the representation from its initial construction. It is suggested that young children's difficulty with reversed sequences may be attributed to a reluctance to reorganize existing representations, rather than to the absence of applicable temporal principles.

Attention↗
PubMed1998

[Isolation of novel expression sequences of C2H2 type zinc finger protein gene from human brain tissue according to the conservation of zinc finger motif].

By low stringency PCR amplification of genomic DNA using the primers designed based on the conservation of zinc finger motif, we got 8 gradient eletrophoretic bands. After recovery of the second and third bands, the DNA fragments in them were cloned and sequenced. Compared to the GenBank database, among these 60 segments containing zinc finger motif, 23 segments were novel zinc finger genes' genomic segments. Then the human brain tissue cDNA library was screened, using these segments as probes, and 44 positive clones were obtained. Rescreening 28 of them, we got 20 rescreened clones. All of them were sequenced and sent to the GenBank DNA database for sequence analysis, the results showed that 16 were novel C2H2 type zinc finger protein cDNA segments. The cDNA segments encoding the novel C2H2 type zinc finger proteins provide the basic materials for cloning of full length cDNA of valuable novel zinc finger protein genes.

Amino Acid Sequence↗
PubMed2005

A novel HLA-B*420502 allele identified by PCR-SSO/SSP routine typing and confirmed by Sequencing-based typing.

A novel human leukocyte antigen-B (HLA-B) allele, B*420502, was identified in a patient with leukemia (Caucasoid, Czech ancestry) and his mother during intrafamily search for the hematopoietic stem cell donor. The novel allele was initially detected by HLA typing at low resolution using both sequence specific primers and sequence specific oligonucleotides techniques that resulted in unique reaction patterns. The alleles of the HLA-B locus were separated by the haplotype-specific extraction technique. Sequencing of those alleles revealed a novel allele, B*420502, that is identical with B*420501 except a T-->G exchange (synonymous mutation) at position 618.

Alleles↗
PubMed1999

Sequencing of HLA class II genes based on the conserved diversity of the non-coding regions: sequencing based typing of HLA-DRB genes.

In this paper, we present a novel sequencing based typing strategy for the HLA-DRB1, 3, 4 and 5 loci. The new approach is based on a group-specific amplification from intron 1 to intron 2 according to the serologically-defined antigens. For this purpose, we have determined the 3' 500 bp-fragment of intron 1 and the 5' 340 bp-fragment of intron 2 of all serological antigens and their most frequent subtypes. We discovered a remarkably conserved diversity characterized by lineage-specific sequence motifs. This lineage-specificity of non-coding motifs in the 1st and 2nd intron offered the possibility to establish a clear serology-related amplification strategy. The method allows the complete analysis of the 2nd exon and the definition of the cis/trans linkage of sequence motifs by intron-mediated polymerase chain reaction (PCR)-based separation of the haplotypes in nearly all serologically heterozygous samples. In particular, the non-coding variabilities between the DR52-associated DRB1 groups made their independent amplification possible. Thus, compared to the standard procedures using exon-based amplification primers, the groups DR3, DR12, some DR13 alleles (1301, 1302) and the DR14 group could be amplified by specific primer mixes. The DR8 could be amplified with an individual primer mix not co-amplifying the DR12. The DR11 and DR13 did not show any individual motif in intron 1 or intron 2. In order to achieve a separate amplification, they had to be amplified by multispecific primer mixes (DR3/11/13/14; DR3/11/13 or DR11/13/14) excluding the other haplotype. Thus, exclusively the alleles in rare DR11,13 heterozygosities without a DRB1*1301 or 1302 could not be amplified separately. Fourteen primer mixes are used to amplify the specificities DR1-14, and 6 primer mixes for the specificities DR51-53. The sequence homology of the 3' end of intron 1 facilitated the application of only three different sequencing primers for all DRB alleles.

Alleles↗