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PubMed1992

Identification of transcriptional regulatory activity within the 5' A-type monomer sequence of the mouse LINE-1 retroposon.

LINE-1 (L1) is a retroposon found in all mammals. In the mouse, approximately 10% of L1 elements are full-length and can be grouped into two classes, A or F, based upon the type of monomer sequence repeated at the 5' end. In order to test for promoter activity in the 5' end of the A-type mouse L1 element, we cloned several different A-monomers into a promoterless chloramphenicol acetyltransferase (CAT) vector. The A-monomer constructs varied in their ability to regulate transcription of the CAT gene, exhibiting CAT activity 16-37% of that detected with the Rous sarcoma virus promoter and enhancer. A series of A-monomer deletions were tested for their ability to regulate CAT expression and gel retardation experiments were performed to identify regions of the A-monomer that may be involved in L1 transcriptional regulation. A-monomer sequences are usually found repeated 2-5 times at the 5' end of a full-length mouse L1. In the absence of long terminal repeats or an internal promoter, the tandem array of A-monomers may provide a mechanism for A-type L1 elements to generate transcripts containing transcriptional regulatory sequences.

Animals↗
PubMed1996

Arginine 75 in the pseudosubstrate sequence of type Ibeta cGMP-dependent protein kinase is critical for autoinhibition, although autophosphorylated serine 63 is outside this sequence.

Autoinhibitory domains in many protein kinases include either a phosphorylatable substrate-like sequence or a pseudosubstrate sequence. This study shows that Ibeta cGMP-dependent protein kinase (cGK) autophosphorylates Ser-63, which is in an atypical cGK substrate sequence (-59AQKQAS-) that is amino-terminal to the pseudosubstrate motif (-74KRQAI-). cGMP increases the rate of autophosphorylation (approximately 0.8 phosphate/cGK monomer) approximately 3-fold. Autophosphorylation is an intramolecular process since it is independent of cGK concentration. cGMP activation of cGK enhances proteolysis within and near the pseudosubstrate site; treatment of dimeric cGK with three proteases produces three cGK monomers (approximately 67-70 kDa each). Their amino-terminal sequences are 75RQAISAEPT-, 76QAISAEPTAF-, and 86DIQDLSXV-, respectively. cGMP stimulates these kinases by 10-, 2.5-, and 1.4-fold, respectively, compared with a 10-fold effect on intact cGK. Increased basal activity accounts for the diminished stimulation. Thus, the primary autophosphorylation site of Ibeta cGK is well outside the pseudosubstrate site, but Arg-75 in the pseudosubstrate site is critical for autoinhibition. Autoinhibition also involves elements that are carboxyl-terminal to Arg-75.

Amino Acid Sequence↗
PubMed2025

Transmission of extended spectrum β-lactamase-producing Escherichia coli and antimicrobial resistance gene flow across One Health compartments in eastern Africa: a whole-genome sequence analysis from a prospective cohort study.

BACKGROUND: The One Health paradigm considers interdependence of human, animal, and environmental health. However, there is little evidence from high-income countries to support the importance of a One Health approach to addressing spread of antimicrobial resistance (AMR). Given AMR is a global threat, understanding how the close interactions of humans with animals and the environment in low-income settings affect the spread of AMR is important. We aimed to investigate diversity and transmission of extended spectrum β-lactamase (ESBL)-producing Escherichia coli across household-linked One Health compartments using genomic data. METHODS: We sequenced whole genomes of ESBL-producing E coli isolates from humans, animals, and the environment from a prospective, longitudinal cohort study conducted in Malawi (April 29, 2019, to Dec 3, 2020) and Uganda (July 16, 2020, to Aug 6, 2021). In the cohort study, 259 households were enrolled at baseline in Malawi and 92 in Uganda from a mix of urban, peri-urban, and rural areas. Households were followed up at months 1, 3, and 6 in Malawi and at months 1, 2, and 4 in Uganda. Samples collected at each visit included human and animal stool, environmental samples from hand-contact areas, food, and water, and broader environmental samples such as river water. Samples were cultured in buffered peptone water and then ESBL chromogenic agar to isolate ESBL-producing E coli. ESBL-producing E coli isolates underwent whole-genome sequencing. We performed phylogenetic analyses, and in-silico multi-locus sequence typing, characterised AMR determinants and linked genotypes with sample location, ecological source, and other covariates. We performed fine-scale single nucleotide polymorphism (SNP) and network analysis to infer strain and plasmid transmission across ecological compartments. The primary outcome was colonisation with ESBL-producing E coli. Secondary outcomes were genomic clusters and ESBL genomic determinants within and between One Health compartments. FINDINGS: We found high diversity of ESBL-producing E coli, with 170 sequence types and 166 genomic clusters identified from 2344 genomes, including 1814 genomes from Malawi (907 human, 221 animal, and 686 environmental) and 530 genomes from Uganda (380 human, 147 animal, and three environmental). Sequence type (ST)131 dominated in Malawi (209 [11·5%] of 1814 genomes), and ST10 dominated in Uganda (45 [8·5%] of 530 genomes). Common ESBL genes blaCTX-M-15 (1604 [68·4%] of 2344 genomes) and blaCTX-M-27 (336 [14·3%] of 2344 genomes) were carried on a complex network of 55 and 30 different plasmids. This diversity of plasmids presented multiple pathways for dissemination and revealed high force of selection. Phylogenetic analyses revealed common intermixing of isolates between humans, animals, and the environment. SNP transmission analysis revealed ecologically overlapping clusters, suggesting ESBL-producing E coli co-circulation both within and between compartments with frequent spillover events. Applying a five-SNP threshold, we inferred 463 human-environment transmission events, 146 human-animal events, and 142 animal-environment events. INTERPRETATION: Our work suggests that a One Health approach is crucial to addressing AMR in eastern Africa. Improving water, sanitation, and hygiene systems will create a safer environment, reduce spillovers of AMR bacteria between compartments, and eventually reduce AMR reservoirs in the environment and in animals. FUNDING: Medical Research Council, National Institute for Health and Care Research, and Wellcome Trust.

Humans↗
PubMed1993

Characterization of the genomic sequence of type V (or 3a) hepatitis C virus isolates and PCR primers for specific detection.

We have identified four new hepatitis C virus (HCV) isolates whose genomic RNA could be amplified by PCR using primers from the 5' untranslated region (UTR), but the RNA could not be detected with genotype I to IV (or types 1a, 1b, 2a and 2b respectively)-specific core region-derived primers. We compared the nucleotide sequences of the new isolates from positions 65 to 1850 (3' end of 5' UTR, C, E1 and 5' end of E2/NS1) and 8276 to 9394 (3' end of NS5 and 3' UTR) with those for genotypes I to IV. The four isolates had the following characteristics: (i) the overall nucleotide sequence similarity between the four isolates was 95 to 96%, compared to 73 to 74%, 73%, 70% or 69 to 70% against genotypes I, II, III or IV, respectively; (ii) the sequence similarity to other reported 'type V (3a)' isolates was 88 to 100%; (iii) the hypervariable region 1 [(HVR)-1] was present but HVR-2 was absent within the E2/NS1 region; (iv) only one in-frame termination codon was present for the presumed polyprotein; (v) the 3'UTR preceding a terminal poly(U) stretch was significantly shorter than in genotype I to IV isolates. We classified the four isolates as genotype V (3a), and searched for uniquely conserved nucleotide sequences that could be used for type-specific PCR. A core region-derived primer pair (no. 104V: 5' CGTAAAACTTCT GAACGGTC, sense and no. 339: 5' GCTGAGCCCA GGACCGGTCT, antisense) was identified and successfully used to diagnose genotype V (3a) HCV infection.

Amino Acid Sequence↗
PubMed1992

Sensitivity of the polymerase chain reaction for detecting human T-cell leukemia virus type I sequences in paraffin-embedded tissue. Effect of unbuffered formalin fixation.

Recently, the application of the polymerase chain reaction (PCR) to formalin-fixed paraffin-embedded tissue has been reported. But formalin, especially unbuffered formalin, is known to break DNA into small fragments. DNA extracted from MT-2 cells fixed in unbuffered formalin for various periods of time were subjected to the PCR and the effect of unbuffered formalin fixation on the ability of the PCR to detect exogenous sequences; i.e., human T-cell leukemia virus type I (HTLV-I) proviral DNA, was examined. The sensitivity of the PCR decreased as a function of both the duration of fixation and the length of the expected DNA products. When the expected length of the PCR product was about 200 bp, a slight decrease in the sensitivity was observed after 4-day fixation. When it was about 300 bp, a similar decrease was observed following 4-h fixation. In the case of a 500 bp product, the sensitivity began to decrease after 30-min fixation and a 100-fold decrease was observed after 10-day fixation. A decrease was not observed, however, with a 100 bp product. The appropriate design of primers, especially with regard to the length of the amplified product, is essential to keep the sensitivity of the PCR, particularly when the target tissues have been fixed in unbuffered formalin.

Base Sequence↗
PubMed1996

A computerized method to predict the discriminatory properties for class II sequencing based typing.

Accurate identification of alleles or groups of alleles is obtained using DNA techniques for HLA class II typing. In PCR-SSP and PCR-SSO the typing resolution is dependent on the location of the amplification primers and the number of oligonucleotide probes used for hybridization. These primers are developed based upon the known allele sequences. However, the number of published allele sequences increases rapidly. Periodic reconsideration of the discriminatory properties of the methods used is necessary and often leads to the addition of primers, and an increase of ambiguous genotypes. SBT is a high-resolution class II typing method which utilizes the second exon sequence at all positions for typing. Using a generic amplification and sequencing approach, a limited number of ambiguous heterozygotes can be expected. However, the number of ambiguities is dependent on the location of the primers. These ambiguities can be solved by the use of group-specific primers in either the amplification or sequencing reaction. To identify the applicability of a SBT approach with respect to typing resolution, it is important to identify these ambiguities and develop strategies to solve them. We developed software that predicts the ambiguous heterozygotes, and analyzes their sequences for group specific primers, solving these ambiguities. The location of the primers is considered as well as the use of generic vs group-specific amplification or sequencing reactions. Allele sequences of all class II genes, as accepted by the WHO nomenclature committee for factors of the HLA system and submitted to the EMBL/Genbank databases, are analyzed to define the allelic discrimination level of HLA-DQB1, -DQA1, -DFB1, -DPA1, -DRB1, -DRB3, -DRB4, and -DRB5.

Alleles↗
PubMed1997

Vertical distribution and phylogenetic characterization of marine planktonic Archaea in the Santa Barbara Channel.

Newly described phylogenetic lineages within the domain Archaea have recently been found to be significant components of marine picoplankton assemblages. To better understand the ecology of these microorganisms, we investigated the relative abundance, distribution, and phylogenetic composition of Archaea in the Santa Barbara Channel. Significant amounts of archaeal rRNA and rDNA (genes coding for rRNA) were detected in all samples analyzed. The relative abundance of archaeal rRNA as measured by quantitative oligonucleotide hybridization experiments was low in surface waters but reached higher values (20 to 30% of prokaryotic rRNA) at depths below 100 m. Probes were developed for the two major groups of marine Archaea detected. rRNA originating from the euryarchaeal group (group II) was most abundant in surface waters, whereas rRNA from the crenarchaeal group (group I) dominated at depth. Clone libraries of PCR-amplified archaeal rRNA genes were constructed with samples from 0 and 200 m deep. Screening of libraries by hybridization with specific oligonucleotide probes, as well as subsequent sequencing of the cloned genes, indicated that virtually all archaeal rDNA clones recovered belonged to one of the two groups. The recovery of cloned rDNA sequence types in depth profiles exhibited the same trends as were observed in quantitative rRNA hybridization experiments. One representative of each of 18 distinct restriction fragment length polymorphism types was partially sequenced. Recovered sequences spanned most of the previously reported phylogenetic diversity detected in planktonic crenarchaeal and euryarchaeal groups. Several rDNA sequences appeared to be harbored in archaeal types which are widely distributed in marine coastal waters. In total, data suggest that marine planktonic crenarchaea and euryarchaea of temperate coastal habitats thrive in different zones of the water column. The relative rRNA abundance of the crenarchaeal group suggests that its members constitute a significant fraction of the prokaryotic biomass in subsurface coastal waters.

Animals↗
PubMed2026

Multidrug resistance and genomic characteristics of nontypeable Haemophilus influenzae isolates from the respiratory tract of pediatric patients.

UNLABELLED: Nontypeable Haemophilus influenzae (NTHi) is a common colonizer of the human upper respiratory tract and one of the major pathogens responsible for pediatric respiratory tract infections. Given the increasing severity of its multidrug resistance (MDR), this study comprehensively investigated the genomic characteristics of circulating NTHi isolated from sputum and bronchoalveolar lavage fluid (BALF). A total of 104 H. influenzae isolates (69 from sputum; 35 from BALF) were collected from pediatric patients between January 2024 and January 2025. All isolates underwent whole-genome sequencing and antimicrobial susceptibility testing, followed by core/pan-genome phylogenetic analysis, multilocus sequence typing (MLST), and resistome profiling. Among them, 103 were identified as NTHi. We identified 29 known sequence types (STs) and 10 novel STs, with ST-107 (14.4%), ST-57 (10.6%), and ST-11 (8.7%) being the major circulating lineages. However, core-genome phylogenetic analysis provided a more granular view of the genetic variation within these identical STs. All the isolates showed high resistance to ampicillin (98.1%) and cefuroxime (84.6%). Genomically, the multidrug efflux pump gene hmrM was ubiquitous (100%). Ampicillin resistance was predominantly driven by blaTEM-1 carriage (77.9%), with minor contributions from chromosomal ftsI mutations. Fifteen plasmid replicons were predicted from 25 isolates, which highly coincided with the carriage of blaTEM-1 and other acquired resistance genes. This study demonstrates that MDR in pediatric NTHi is primarily driven by acquired resistance genes and chromosomal mutations, with specific resistant clones persisting and enriching under clinical antibiotic pressures. These findings underscore the importance of continuous high-resolution genomic surveillance in guiding rational antibiotic stewardship. IMPORTANCE: This study highlights the critical importance of high-resolution genomic surveillance in managing pediatric nontypeable Haemophilus influenzae (NTHi) infections. By utilizing whole-genome sequencing, we uncovered the pathogen's highly dynamic population structure and complex multidrug resistance (MDR) mechanisms. Crucially, our findings reveal a strong, non-random coupling between core genomic architectures, virulence factors, and MDR elements, driven by dual environmental and pharmacological pressures. This "virulence-MDR" co-evolutionary trend underscores the persistent clinical threat of locally adapted high-risk clones. These findings provide important insights for guiding rational clinical antibiotic stewardship, optimizing treatment strategies, and improving regional infection control.

Humans↗
PubMed1983

Comparative structure and evolution of goat and sheep satellite I DNAs.

The satellite I DNAs of domestic goat (Capra hircus) and domestic sheep (Ovis aries) have been studied using molecular hybridisation and restriction enzyme analysis. Both satellite DNAs are composed of repeat units of 820 base pairs in length, but their restriction maps, although similar, differ in certain respects. Thus the majority of sheep satellite I repeat units have two EcoRI sites and one AluI site, whereas the majority of goat satellite I repeat units have one EcoRI site and two AluI sites. The sheep satellite I repeat units with the two EcoRI sites are much more homogeneous than the repeats forming the remainder of the satellite, as judged by the difference in the melting temperatures of native and reassociated molecules. DNAs from species of wild sheep and goats were screened for the presence of these repeat units, and they appear to have been amplified during the radiation of the Ovis genus. Goat satellite I is composed of a single sequence type which has changed through base substitution until the sequence now shows considerable heterogeneity. It is proposed that the major sequence types of these two satellite DNAs were amplified by different saltatory replication events at different times in the evolution of the group.

Animals↗
PubMed1993

Conservation of motifs within the unusually variable polypeptide sequences of type I restriction and modification enzymes.

Type I restriction enzymes comprise three subunits encoded by genes designated hsdR, hsdM, and hsdS; S confers sequence specificity. Three families of enzymes are known and within families, but not between, hsdM and hsdR are conserved. Consequently, interfamily comparisons of M and R sequences focus on regions of putative functional significance, while both inter- and intrafamily comparisons address the origin, nature and role of diversity of type I restriction systems. We have determined the sequence of the hsdR gene for EcoA, thus making available sequences of all three hsd genes of one representative from each family. The predicted R polypeptide sequences share conserved regions with one superfamily of putative helicases, so-called 'DEAD box' proteins; these conserved sequences may be associated with the ATP-dependent translocation of DNA that precedes restriction. We also present hsdM and hsdR sequences for EcoE, a member of the same family as EcoA. The sequences of the M and R genes of EcoA and EcoE are at least as divergent as typical genes from Escherichia coli and Salmonella, perhaps as the result of selection favouring diversity of restriction specificities combined with lateral transfer among different species.

Amino Acid Sequence↗
PubMed2026

An analysis of Wolbachia incidence and genetics in non-ant Hymenoptera diversity.

Wolbachia bacteria are widespread maternally inherited symbionts of Nematoda and diverse Arthropoda hosts. Their evolutionary success is determined by the ability to affect the biology of the host in different ways, promoting the relative fitness of females harbouring Wolbachia, as well as sporadic cases of horizontal transmission of Wolbachia between different host species. Here, we revised Wolbachia infection in the Hymenoptera with respect to the symbiont occurrence in host taxa and Wolbachia genetics. The representatives of about half of the extant families and 1000 out of 140,000 non-ant hymenopteran species have been tested for Wolbachia infection. We concluded that Wolbachia are found in all major hymenopteran families. More than 75% of Wolbachia diversity belongs to the A supergroup, whereas other variants belong to the B supergroup and only two isolates belong to the supergroup F. One of the main results of this study is the discovery of a specific Wolbachia genetic pattern (based on multilocus sequence typing [MLST]) in Apoidea hosts. Two haplotypes, ST-479 and ST-wH14, along with their alleles within other sequence types (STs), form the core of symbiont diversity, comprising 81% of unique host-Wolbachia ST associations. These haplotypes have not been reported beyond the Apoidea superfamily or Hymenoptera order. The reasons and mechanisms underlying this pattern in Apoidea remain unknown. Another important result of our study concerns the use of the MLST protocol, which has been previously criticised. We analysed 51 Wolbachia genomes for the average nucleotide identity (ANI) and MLST data, and found that genome and MLST variation are highly correlated. Therefore, the MLST protocol for Wolbachia remains reliable for many research tasks.

Animals↗
PubMed1993

A G-->A substitution in an HNF I binding site in the human alpha-fetoprotein gene is associated with hereditary persistence of alpha-fetoprotein (HPAFP).

A family displaying hereditary persistence of alpha-fetoprotein (HPAFP) in adult life was detected in an antenatal screening programme for spina bifida. RFLP linkage analysis shows that the trait is linked with the albumin-AFP locus. The molecular mechanism responsible for the post-natal repression of the AFP gene is unknown. We wished to determine the molecular mechanism underlying HPAFP in this family. Sequence analysis of the 5'-flanking sequences of their gene revealed a GA substitution at position -119 associated with the trait. This substitution occurs in a potential HNF I binding site, and increases the similarity of the sequence to a consensus HNF I recognition site. In a competitive gel retardation assay the mutant sequence binds HNF I alpha more tightly than the wild type sequence. Furthermore, 5'-flanking sequences of the human AFP gene containing the G-->A substitution direct a higher level of CAT expression in transfected human hepatoma cells than the wild type sequences. We conclude that the G-->A substitution at position -119 of the AFP gene is the mutation causing HPAFP in this family. These results highlight the importance of this HNF I binding site in the developmental regulation of the AFP gene.

Adult↗
PubMed1996

Action at a distance in supercoiled DNA: effects of sequence on slither, branching, and intramolecular concentration.

We report a computer modeling study of DNA supercoiling in model plasmids over the size range of 140-1260 bp. We used a computer model with basepair resolution. Molecular dynamics was used to produce ensembles at 300 K and to investigate intramolecular motions. The plasmid models varied by their sequence. The sequence types employed for comparison included a curve-bearing plasmid, a heterogenous sequence plasmid, and a homogenous sequence. Within the three sequence types tested at the 1260-bp plasmid size, we observed several sequence-dependent phenomena. Writhe, radius of gyration, slither motion, and branching probability were seen to be sequence dependent. Branching probability was the least in the homogenous plasmid and the greatest in the curve-bearing plasmid. The curve imposed a symmetry on the plasmid that was absent in the heterogenous sequence. Significant localizations and enhancements of intramolecular concentration were seen to a persistence length. Molecular dynamics allowed us to observe the mechanism of branch formation and reabsorption. We observed a size-dependent change in the types of motion observed in plasmids. Slither motion predominated in plasmids up to 600 bp in size, whereas global rearrangements were more important in the 1260 mer.

Base Sequence↗
PubMed2026

Characteristics of an NDM-1-producing Klebsiella pneumoniae strain belonging to ST105.

Uncommon multilocus sequence types (MLSTs) of NDM-producing Klebsiella pneumoniae may pose a significant threat to patients, although they are often overlooked in surveillance studies. Characterizing these isolates is therefore important for infection control. In this study, the antimicrobial susceptibility and pathogenicity of K. pneumoniae strain KP_WXD, pertaining to the atypical sequence type ST105, were evaluated, including capsular polysaccharide (CPS) production, biofilm formation, and resistance to serum killing. Whole-genome sequencing (WGS) was performed to analyze its genomic features. K. pneumoniae KP_WXD strain was resistant to all tested β-lactam agents. Its virulence was lower than that of K. pneumoniae strains ST11-KL64 and NTUH-k2044 used as references, while its biofilm formation ability was stronger than that of both strains. WGS analysis revealed carriage of IncF and IncN plasmids carrying multiple antibiotic resistance genes, alongside blaNDM-1 and blaCTX-M, integrated into well-characterized mobile genetic elements. Moreover, both blaNDM-1 and blaCTX-M-harboring plasmids were transferable to E. coli J53 by conjugation without significant fitness cost on the recipient strain.

Klebsiella pneumoniae↗
PubMed1996

Analysis of position of trial sequence and type of diluent on the detection threshold for phenyl ethyl alcohol using a single staircase method.

Although detection thresholds for odors are commonly measured in academic and medical settings, the influences of procedural factors on threshold values are poorly understood. The present study evaluated the influences of (i) trial sequence position and (ii) diluent type on the threshold value for the rose-like odorant phenyl ethyl alcohol. In Exp. 1, detection thresholds were measured in 24 subjects on two occasions in which different diluents were used in the concentration series, propylene glycol and light mineral oil. The thresholds were estimated using a 7-reversal initially ascending single-staircase procedure. Threshold values were significantly influenced by the type of diluent (lower for mineral oil) and trial sequence (lower for later threshold reversals). In Exp. 2, 24 subjects were administered a staircase threshold test which continued through 15 staircase reversals. Continued testing resulted in a significant lowering of the threshold measure. These findings demonstrate the importance of both diluent and test length on detection threshold values measured by a single staircase procedure and emphasize the need for standardization of procedures for threshold testing.

Adolescent↗
PubMed1987

Structural and transcriptional analysis of human papillomavirus type 16 sequences in cervical carcinoma cell lines.

We cloned and analyzed the integrated human papillomavirus type 16 (HPV-16) genomes that are present in the human cervical carcinoma cell lines SiHa and CaSki. The single HPV-16 genome in the SiHa line was cloned as a 10-kilobase (kb) HindIII fragment. Integration of the HPV-16 genome occurred at bases 3132 and 3384 with disruption of the E2 and E4 open reading frames (ORFs). An additional 52-base-pair deletion of HPV-16 sequences fused the E2 and E4 ORFs. the 5' portion of the disrupted E2 ORF terminated immediately in the contiguous human right-flanking sequences. Heteroduplex analysis of this cloned integrated viral genome with the prototype HPV-16 DNA revealed no other deletions, insertions, or rearrangements. DNA sequence analysis of the E1 ORF, however, revealed the presence of an additional guanine at nucleotide 1138, resulting in the fusion of the E1a and E1b ORFs into a single E1 ORF. Sequence analysis of the human flanking sequences revealed one-half of an Alu sequence at the left junction and a sequence highly homologous to the human O repeat in the right-flanking region. Analysis of the three most abundant BamHI clones from the CaSki line showed that these consisted of full-length, 7.9-kb HPV-16 DNA; a 6.5-kb genome resulting from a 1.4-kb deletion of the long control region; and a 10.5-kb clone generated by a 2.6-kb tandem repeat of the 3' early region. These HPV-16 genomes were arranged in the host chromosomes as head-to-tail, tandemly repeated arrays. Transcription analysis revealed expression of the HPV-16 genome in each of these two cervical carcinoma cell lines, albeit at significantly different levels. Preliminary mapping of the viral RNA with subgenomic strand-specific probes indicated that viral transcription appeared to be derived primarily from the E6 and E7 ORFs.

Amino Acid Sequence↗
PubMed1992

Extrachromosomal human immunodeficiency virus type 1 sequences are methylated in latently infected U937 cells.

Long-term human immunodeficiency virus type 1 (HIV-1) infection of the human monocytic cell line U937 resulted in a progressive loss of infectivity that was correlated with the accumulation of stable, extrachromosomal forms of viral DNA. Viral latency was also characterized by reduced levels of HIV-1 transcription. The structure and activity of extrachromosomal viral DNA (E-DNA) in a fully latent U937 cell line was investigated by molecular cloning and DNA transfection. The resulting 18-kb E-DNA clone was composed of an intact HIV-1 sequence flanked by 7 kb of host sequence to one side and 1 kb of host DNA to the other side. This configuration is the result of retroviral integration into a highly repetitive element of the Alu family. Transfection of the E-DNA clone resulted in the production of infectious virus, indicating that viral latency was not the result of mutations in the HIV-1 genome. Analysis of CCGG sites revealed extensive de novo methylation of viral sequences present within E-DNA. These results suggest that modification of extrachromosomal viral DNA sequences is a mechanism for HIV-1 latency in long-term infected U937 cells.

Base Sequence↗
PubMed2026

Phylogenomic and Clinical Perspectives of an East Asia-Specific Cluster of Mycobacterium massiliense.

BACKGROUND: Mycobacterium abscessus subspecies massiliense (MAM) can form genetically related clusters through continuous within-host adaptations. RESEARCH QUESTION: What is the epidemiologic and clinical significance of the sequence type 120 (ST120) strain of MAM, an East Asia-specific cluster? STUDY DESIGN AND METHODS: Isolates were obtained from patients with MAM pulmonary disease at Seoul National University Hospital between October 1, 2019, and December 31, 2023. These isolates were analyzed using multilocus sequence typing and colony morphotyping and were evaluated for glycopeptidolipid biosynthesis-related gene deletions. Whole-genome sequencing was performed for phylogenomic and pangenome analyses, incorporating MAM genome data from public databases. Finally, the clinical course and treatment outcomes of patients infected with ST120 were evaluated. RESULTS: Among the isolates obtained from 136 patients, 50 isolates (36.8%) were classified as ST120. All ST120 isolates exhibited a rough colony morphotype and harbored deletions in glycopeptidolipid biosynthesis-related genes. From 796 global strains, ST120 was identified exclusively in the isolates obtained from South Korea (51/137 isolates [37.1%]), Japan (3/53 [5.7%]), and Taiwan (2/52 [3.8%]). Genomic analysis revealed the stepwise genetic evolution of ST120, suggesting transmission from Taiwan to Japan and from Japan to South Korea. The ST120 strains exhibited genetic mutations associated with virulence and structural alterations within the ESX system. Consequently, patients with ST120 strain infections had a higher prevalence of cavitary disease (23/50 [46%]) than those infected with non-ST120 strains (19/86 [22.1%]; P = .004). Among those who initiated treatment, the proportion of microbiological cure was lower in patients with ST120 infection (6/15 [40.0%]) than in those with non-ST120 infection (24/34 [70.6%]; P = .043). INTERPRETATION: Our results show that ST120, a strain predominantly found in South Korea, is characterized by the extensive loss of glycopeptidolipid biosynthesis-related genes and is associated with increased disease severity and worse treatment outcomes.

Humans↗