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PubMed1996

Functional dissection of the mouse Hox-a5 gene.

The Hox genes are clustered in evolutionarily conserved complexes and encode DNA binding proteins that determine positional identity. Ubiquitous expression of fly or mammalian Hox proteins in Drosophila embryos provides an assay for gene function, since different Hox genes induce characteristic homeotic transformations. Drosophila Sex combs reduced (Scr) and its murine cognate Hox-a5 produce identical transformations in transgenic flies. To study the contributions of domains conserved between the two proteins, truncated versions of mouse Hox-a5 were assayed for their ability to activate transcription in cultured cells and to induce homeotic transformation and activate target gene expression in transgenic embryos. The homeodomain is essential for protein function and/or nuclear targeting; the N-terminal region contributes to transcription activity and transformation potential in the embryo, but plays no role in determining functional specificity. The YPWM motif is essential for biological specificity, although it does not contribute to transcriptional activation potential. It was recently shown that the Hox-a5 YPWM motif is necessary for in vitro interactions with the co-factor Pbx1. Our results suggest that this type of protein-protein interaction may be essential for the biological activities of Hox-a5 and Scr.

Amino Acid Sequence↗
PubMed2006

Targeted gene expression in the zebrafish prechordal plate.

Targeted gene expression is a powerful tool for understanding gene function in vivo. In zebrafish, overexpression of gene products is typically accomplished ubiquitously, without temporal and spatial specificity. However, the yeast Gal4/UAS system can be used for targeted gene expression in zebrafish. Here we describe the generation and characterization of Tg[gsc: Gal4-VP16] transgenic zebrafish lines that harbor a construct encoding Gal4-VP16 transcriptional activator under the control of a fragment of the goosecoid gene promoter. Tg[gsc:Gal4-VP16] embryos express Gal4-VP16 RNA in presumptive prechordal plate mesendoderm during late blastula and throughout gastrulation. By crossing these fish to Tg[UAS-GFP] transgenic fish, we show that the gsc:Gal4-VP16 transgene is capable of driving strong expression of a target gene in the prechordal plate and its derivatives during gastrulation and segmentation. Thus, the use of Tg[gsc:Gal4-VP16] fish can help in understanding gene function in the prechordal plate, an embryonic structure that is crucial for normal neural patterning.

Animals↗
PubMed2001

Regulation of the muscle-specific expression and function of an ascidian T-box gene, As-T2.

The Tbx6 T-box genes are expressed in somite precursor cells of vertebrate embryos and are essential for the differentiation of paraxial mesoderm. However, it is unclear how spatial regulation of the gene expression is controlled and how the genes function to promote muscle differentiation. The Tbx6-related gene As-T2 of the ascidian Halocynthia roretzi is first expressed very transiently in endodermal cells around the 32- approximately 44-cell stage, is then expressed distinctly and continuously in muscle precursor cells, and later in epidermal cells situated in the distal tip region of the elongating tail. We now show that inhibition of As-T2-mediated transcriptional activation by microinjection of As-T2/En(R) into one-cell embryos resulted in suppression of the expression of the muscle-specific actin gene (HrMA4) and myosin heavy chain gene (HrMHC), but the injection did not affect the differentiation of endodermal cells or tail tip cells, suggesting that the primary function of As-T2 is associated with muscle cell differentiation. The 5' flanking region of As-T2 contains two promoter modules that regulate its specific expression: a distal module that responsible for its specific expression in the tail, and a proximal module required for its muscle-specific expression. Around the proximal module, there are two putative T protein-binding motifs (TTCACACTT). Co-injection of an As-T2/lacZ construct with or without the T-binding motifs together with As-T2 mRNA revealed that these motifs are essential for autoregulatory activation of the gene itself. In addition, we found that the minimal promoter regions of HrMA4 and HrMHC contain T-binding motifs. Co-injection of HrMA4/lacZ or HrMHC/lacZ containing the T-binding motifs along with As-T2 mRNA revealed that As-T2 protein binds to these motifs to upregulate the gene activity. Taking into account the recent finding of maternal molecules for muscle differentiation, we propose a model for a genetic cascade that includes As-T2 as a regulator of muscle cell differentiation in the ascidian embryo.

Actins↗
PubMed2005

Expression profiling reveals functionally important genes and coordinately regulated signaling pathway genes during in vitro angiogenesis.

Angiogenesis is a complex multicellular process requiring the orchestration of many events including migration, alignment, proliferation, lumen formation, remodeling, and maturation. Such complexity indicates that not only individual genes but also entire signaling pathways will be crucial in angiogenesis. To define an angiogenic blueprint of regulated genes, we utilized our well-characterized three-dimensional collagen gel model of in vitro angiogenesis, in which the majority of cells synchronously progress through defined morphological stages culminating in the formation of capillary tubes. We developed a comprehensive three-tiered approach using microarray analysis, which allowed us to identify genes known to be involved in angiogenesis and genes hitherto unlinked to angiogenesis as well as novel genes and has proven especially useful for genes where the magnitude of change is small. Of interest is the ability to recognize complete signaling pathways that are regulated and genes clustering into ontological groups implicating the functional importance of particular processes. We have shown that consecutive members of the mitogen-activated protein kinase and leukemia inhibitory factor signaling pathways are altered at the mRNA level during in vitro angiogenesis. Thus, at least for the mitogen-activated protein kinase pathway, mRNA changes as well as the phosphorylation changes of these gene products may be important in the control of blood vessel morphogenesis. Furthermore, in this study, we demonstrated the power of virtual Northern blot analysis, as an alternative to quantitative RT-PCR, for measuring the magnitudes of differential gene expression.

Bayes Theorem↗
PubMed1998

A novel complex allele and two new point mutations in type 2 (acute neuronopathic) Gaucher disease.

Gaucher disease, the most prevalent inherited sphingolipidosis, is characterized by lipid laden histiocytes in the spleen, liver and bone marrow sinusoids of affected individuals. It results from deleterious mutations in the functional gene of glucocerebrosidase (acid beta-glucosidase, EC. 3.2.1.45) and is transmitted as an autosomal recessive trait. Three clinical forms of Gaucher disease have been described: Type 1 non-neuronopathic, type 2 acute neuronopathic, and type 3 subacute neuronopathic. In this report, we describe the identification and characterization of three novel mutations from two patients who died with type 2 Gaucher disease. Two heterozygous missense point mutations, one at cDNA nucleotide 238A (E41L) and the other at cDNA nucleotide 508T (R131C) were identified, both in the context of a cDNA nucleotide 1448C (L444P) mutation in the second allele. One of these L444P mutations was identified as a novel complex allele resulting from a crossover involving the glucocerebrosidase functional gene and pseudogene beginning between genomic nucleotides 5689 and 5723 and extending through the rest of the coding sequence. Based on the recent identification and sequence analysis of the metaxin gene and pseudogene contiguous with the glucocerebrosidase pseudogene and functional gene respectively, we have developed a PCR-based method for the analysis of the origin and extent of this recombination.

Adult↗
PubMed1993

Characterization of the mouse thrombomodulin gene and functional analysis of the 5' flanking region in F9 teratocarcinoma cells.

The mouse thrombomodulin (TM) gene was examined and shown to be a single copy gene lacking introns. Two different clones each containing the entire mouse TM gene were isolated and the nucleotide sequence of a 1.4 kb fragment comprising the 5' untranslated region and 1.2 kb of flanking sequences was determined. The transcriptional initiation site was located 30 bp downstream from a classical TATA motif within this fragment. This site was used in BALB/c 3T3 cells constitutively expressing TM, and when TM expression was induced in F9 teratocarcinoma cells in response to retinoic acid (RA) and dibutyryl cAMP (dbcAMP). A reporter construct consisting of the 1.4 kb fragment fused to the chloramphenicol acetyl transferase (CAT) gene was used to examine promoter function in F9 cells. CAT activity was induced on exposure to RA and dbcAMP and mimicked the pattern of expression of the endogenous TM gene. Induction of CAT activity did not depend on a sequence resembling a palindromic retinoic acid/thyroid hormone response element. We conclude that the 1.4 kb fragment contains the mouse TM promoter together with elements that control the induction of TM expression in differentiating F9 cells.

Animals↗
PubMed1989

Class II box consensus sequences in the HLA-DR alpha gene: transcriptional function and interaction with nuclear proteins.

The promoter regions of class II major histocompatibility complex genes contain two highly conserved sequences, the X and Y boxes, which may be involved in the control of class II gene expression. In this study, we correlate in vivo functional assays for cis-acting regulatory elements in the HLA-DR alpha gene with in vitro binding assays for trans-acting regulatory proteins. Mutagenesis and transient transfection analyses indicated that both the X and Y boxes were important for HLA-DR alpha promoter function in a B lymphoblastoid cell line. Although specific nuclear protein interactions with the X consensus sequence were not apparent, the Y box, which contained an inverted CCAAT sequence, did bind specifically to at least one nuclear protein. This Y box-binding protein was present in nuclear extracts of all cell types examined, including human B and T cells and HeLa cells. The molecular mass of the protein, as determined by photoactivated protein-DNA cross-linking, was approximately 40 to 50 kilodaltons. Mutagenesis of the Y box that decreased protein binding also decreased promoter activity, implying that protein binding to this DNA sequence is important for DR alpha promoter function.

Base Sequence↗
PubMed1997

Tobacco mutants with a decreased number of functional nia genes compensate by modifying the diurnal regulation of transcription, post-translational modification and turnover of nitrate reductase.

Although nitrate reductase (NR. EC 1.6.6.1) is thought to control the rate of nitrate assimilation, mutants with 40-45% of wildtype (WT) NR activity (NRA) grow as fast as the WT. We have investigated how tobacco (Nicotiana tabacum L. cv. Gatersleben) mutants with one or two instead of four functional nia genes compensate. (i) The nia transcript was higher in the leaves of the mutants. However, the diurnal rhythm was retained in the mutants, with a maximum at the end of the night and a strong decline during the photoperiod. (ii) Nitrate reductase protein and NRA rose to a maximum after 3-4 h light in WT leaves, and then decreased by 50-60% during the second part of the photoperiod and the first part of the night. Leaves of mutants contained 40-60% less NR protein and NRA after 3-4 h illumination, but NR did not decrease during the photoperiod. At the end of the photoperiod the WT and the mutants contained similar levels of NR protein and NRA. (iii) Darkening led to a rapid inactivation of NR in the WT and the mutants. However, in the mutants, this inactivation was reversed after 1-3 h darkness. Calyculin A prevented this reversal. When magnesium was included in the assay to distinguish between the active and inactive forms of NR, mutants contained 50% more activity than the WT during the night. Conversion of [15N]-nitrate to organic compounds in leaves in the first 6 h of the night was 60% faster in the mutants than in the WT. (iv) Growth of WT plants in enhanced carbon dioxide prevented the decline of NRA during the second part of the photoperiod, and led to reactivation of NR in the dark. (v) Increased stability of NR in the light and reversal of dark-inactivation correlated with decreased levels of glutamine in the leaves. When glutamine was supplied to detached leaves it accelerated the breakdown of NR, and led to inactivation of NR, even in the light. (vi) Diurnal changes were also investigated in roots. In the WT, the amount of nia transcript rose to a maximum after 4 h illumination and then gradually decreased. The amplitude of the changes in transcript amount was smaller in roots than in leaves, and there were no diurnal changes in NRA. In mutants, nia transcript levels were high through the photoperiod and the first part of the night. The NRA was 50% lower during the day but rose during the night to an activity almost as high as in the WT. The rate of [15N]-nitrate assimilation in the roots of the mutants resembled that in the WT during the first 6 h of the night. (vii) Diurnal changes were also compared in Nia30(145) transformants with very low NRA, and in nitrate-deficient WT plants. Both sets of plants had similar low growth rates. Nitrate reductase did not show a diurnal rhythm in leaves or roots of Nia30(145), the leaves contained very low glutamine, and NR did not inactivate in the dark. Nitrate-deficient WT plants were watered each day with 0.2 mM nitrate. After watering, there was a small peak of nia transcript NR protein and NRA and, slightly later, a transient increase of glutamine and other amino acids in the leaves. During the night glutamine was low, and NR did not inactivate. In the roots, there was a very marked increase of nitrate, nia transcript and NRA 2-3 h after the daily watering with 0.2 mM nitrate. (viii) It is concluded that WT plants have excess capacity for nitrate assimilation. They only utilise this potential capacity for a short time each day, and then down-regulate nitrate assimilation in response, depending on the conditions, to accumulation of the products of nitrate assimilation or exhaustion of external nitrate. Genotypes with a lower capacity for nitrate assimilation compensate by increasing expression of NR and weakening the feedback regulation, to allow assimilation to continue for a longer period each day.

Circadian Rhythm↗
PubMed2004

Cladosporium fulvum circumvents the second functional resistance gene homologue at the Cf-4 locus (Hcr9-4E ) by secretion of a stable avr4E isoform.

Introgression of resistance trait Cf-4 from wild tomato species into tomato cultivar MoneyMaker (MM-Cf0) has resulted in the near-isogenic line MM-Cf4 that confers resistance to the fungal tomato pathogen Cladosporium fulvum. At the Cf-4 locus, five homologues of Cladosporium resistance gene Cf-9 (Hcr9s) are present. While Hcr9-4D represents the functional Cf-4 resistance gene matching Avr4, Hcr9-4E confers resistance towards C. fulvum by mediating recognition of the novel avirulence determinant Avr4E. Here, we report the isolation of the Avr4E gene, which encodes a cysteine-rich protein of 101 amino acids that is secreted by C. fulvum during colonization of the apoplastic space of tomato leaves. By complementation we show that Avr4E confers avirulence to strains of C. fulvum that are normally virulent on Hcr9-4E-transgenic plants, indicating that Avr4E is a genuine, race-specific avirulence determinant. Strains of C. fulvum evade Hcr9-4E-mediated resistance either by a deletion of the Avr4E gene or by production of a stable Avr4E mutant protein that carries two amino acid substitutions, Phe(82)Leu and Met(93)Thr. Moreover, we demonstrate by site-directed mutagenesis that the single amino acid substitution Phe(82)Leu in Avr4E is sufficient to evade Hcr9-4E-mediated resistance.

Cladosporium↗
PubMed2006

A longitudinal study of the effect of GSTT1 and GSTM1 gene copy number on survival.

Deletions of the glutathione S-transferase superfamily genes GSTT1 and GSTM1 has been associated with oxidative stress related diseases and recently explored as factors implicated in longevity as well. Reported results have been conflicting, which may partially be caused by the traditional use of assays unable to discriminate between carriers of one or two functional genes. Using a quantitative realtime PCR method facilitating quantification of gene copy number, we evaluated the influence of GSTT1 and GSTM1 gene deletions on longevity in a longitudinal study of 681 elderly Danish twins. The mean follow-up time was 7.6 years and during this time a total of 294 deaths occurred. The results demonstrated a non-significant trend for carriage of two copies of the GSTM1 functional gene to be a protective factor, whereas both heterozygosity and homozygosity for the GSTT1 functional gene was associated with a moderate but significant increased mortality in women (hazard rate 2.46 (CI95: 1.43-4.23) and 2.22 (CI95: 1.25-3.94) for one and two alleles, respectively). To our knowledge, this is the first longitudinal study exploring the influence of GST gene polymorphisms on longevity and these data implies that GST gene copy numbers do affect mortality risk in the elderly.

Aged↗
PubMed1995

How often do duplicated genes evolve new functions?

A recently duplicated gene can either fix a null allele (becoming a pseudogene) or fix an (advantageous) allele giving a slightly different function, starting it on the road to evolving a new function. Here we examine the relative probabilities of these two events under a simple model. Null alleles are assumed to be neutral; linkage effects are ignored, as are unequal crossing over and gene conversion. These assumptions likely make our results underestimates for the probability that an advantageous allele is fixed first. When new advantageous mutations are additive with selection coefficient s and the ratio of advantageous to null mutations is rho, the probability an advantageous allele is fixed first is ([1 - e-s]/[rho S] + 1)-1, where S = 4Nes with Ne the effective population size. The probability that a duplicate locus becomes a pseudogene, as opposed to evolving a new gene function, is high unless rhoS >> 1. However, even if advantageous mutations are very rare relative to null mutations, for sufficiently large populations rhoS >> 1 and new gene function, rather than pseudogene formation, is the expected fate of most duplicated genes.

Biological Evolution↗
PubMed2000

A mutated hygromycin resistance gene is functional in the n-alkane-assimilating yeast Candida tropicalis.

Development of a transformation system in the n-alkane-assimilating diploid yeast Candida tropicalis requires an antibiotic resistance gene in order to establish a selectable marker. The resistance gene for hygromycin B has often been used as a selectable marker in yeast transformation. However, C. tropicalis harboring the hygromycin resistance gene (HYG) was as sensitive to hygromycin B as the wild-type strain. Nine CTG codons were found in the ORF of the HYG gene. This codon has been reported to be translated as serine rather than leucine in Candida species. Analysis of the tRNA gene in C. tropicalis with the anticodon CAG [tRNA(CAG) gene], which is complementary to the codon CTG, showed that the sequence was highly similar to that of the C. maltosa tRNA(CAG) gene. In C. maltosa, the codon CTG is read as serine and not leucine. These results suggested that the HYG gene was not functional due to the nonuniversal usage of the CTG codon. Each of the nine CTG codons in the ORF of the HYG gene was changed to a CTC codon, which is read as leucine, by site-directed mutagenesis. When a plasmid containing the mutated HYG gene (HYG#) was constructed and introduced into C. tropicalis, hygromycin-resistant transformants were successfully obtained. This mutated hygromycin resistance gene may be useful for direct selection of C. tropicalis transformants.

Alkanes↗
PubMed2001

Generation of single-copy transgenic mouse embryos directly from ES cells by tetraploid embryo complementation.

BACKGROUND: Transgenic mice have been used extensively to analyze gene function. Unfortunately, traditional transgenic procedures have only limited use in analyzing alleles that cause lethality because lines of founder mice cannot be established. This is frustrating given that such alleles often reveal crucial aspects of gene function. For this reason techniques that facilitate the generation of embryos expressing such alleles would be of enormous benefit. Although the transient generation of transgenic embryos has allowed limited analysis of lethal alleles, it is expensive, time consuming and technically challenging. Moreover a fundamental limitation with this approach is that each embryo generated is unique and transgene expression is highly variable due to the integration of different transgene copy numbers at random genomic sites. RESULTS: Here we describe an alternative method that allows the generation of clonal mouse embryos harboring a single-copy transgene at a defined genomic location. This was facilitated through the production of Hprt negative embryonic stem cells that allow the derivation of embryos by tetraploid embryo complementation. We show that targeting transgenes to the hprt locus in these ES cells by homologous recombination can be efficiently selected by growth in HAT medium. Moreover, embryos derived solely from targeted ES cells containing a single copy LacZ transgene under the control of the alpha-myosin heavy chain promoter exhibited the expected cardiac specific expression pattern. CONCLUSION: Our results demonstrate that tetraploid embryo complementation by F3 hprt negative ES cells facilitates the generation of transgenic mouse embryos containing a single copy gene at a defined genomic locus. This approach is simple, extremely efficient and bypasses any requirement to generate chimeric mice. Moreover embryos generated by this procedure are clonal in that they are all derived from a single ES cell lines. This facilitates the comparative analysis of lethal alleles and thereby advances our ability to analyze gene function in mammals.

Alleles↗
PubMed1992

Chromosomal localization of the murine gene and two related sequences encoding high-mobility-group I and Y proteins.

HMG-I and its isoform HMG-Y are members of the abundant high-mobility-group of nonhistone chromatin proteins; they bind to A + T-rich regions of chromosomal DNA and are expressed at high levels in rapidly dividing, undifferentiated mammalian cells. HMG-I and HMG-Y are alternatively spliced products of a single functional gene, designated Hmgi in the mouse. Here, we report the occurrence of at least three distinct Hmgi-related loci in the mouse. Only one of these loci was present in all of the 10 mouse strains examined; therefore, this locus most likely represents the transcriptionally active, functional gene, Hmgi. Genetic linkage analysis of interspecific and intersubspecific backcrosses showed that Hmgi is located in the t-complex region of mouse Chromosome 17. Two additional Hmgi-related sequences, Hmgi-rs1 and Hmgi-rs2, were found only in certain mouse strains and probably represent pseudogenes. Hmgi-rs1 is located on Chromosome 11; it was present in all of the standard laboratory inbred mouse strains examined but was absent in wild-derived inbred strains of Mus spretus, M. musculus castaneus, and M. m. molossinus. Hmgi-rs2 was found only in M. m. castaneus and is located on Chromosome 6. Hmgi genes have not been previously mapped in any species, but the location of the probable functional gene on murine Chromosome 17 suggests that the homologous gene in humans is located on Chromosome 6.

Animals↗
PubMed2006

Generation of marker- and backbone-free transgenic potatoes by site-specific recombination and a bi-functional marker gene in a non-regular one-border agrobacterium transformation vector.

A binary vector, designated PROGMO, was constructed to assess the potential of the Zygosaccharomyces rouxii R/Rs recombination system for generating marker- and backbone-free transgenic potato (Solanum tuberosum) plants with high transgene expression and low copy number insertion. The PROGMO vector utilises a constitutively expressed plant-adapted R recombinase and a codA-nptII bi-functional, positive/negative selectable marker gene. It carries only the right border (RB) of T-DNA and consequently the whole plasmid will be inserted as one long T-DNA into the plant genome. The recognition sites (Rs) are located at such positions that recombinase enzyme activity will recombine and delete both the bi-functional marker genes as well as the backbone of the binary vector, leaving only the gene of interest flanked by a copy of Rs and RB. Efficiency of PROGMO transformation was tested by introduction of the GUS reporter gene into potato. It was shown that after 21 days of positive selection and using 300 mgl(-1 )5-fluorocytosine for negative selection, 29% of regenerated shoots carried only the GUS gene flanked by a copy of Rs and RB. The PROGMO vector approach is simple and might be widely applicable for the production of marker- and backbone-free transgenic plants of many crop species.

Gene Dosage↗
PubMed2006

Novel gene ashwin functions in Xenopus cell survival and anteroposterior patterning.

The novel gene ashwin was isolated in a differential display screen for genes activated or up-regulated early in neural specification. ashwin is expressed maternally and zygotically, and it is up-regulated in the neural ectoderm after the midgastrula stage. It is expressed in the neural plate and later in the embryonic brain, eyes, and spinal cord. Overexpression of ashwin in whole embryos leads to anterior truncations and other defects. However, a second Organizer does not form, and the secondary axial structures may result from splitting of the Organizer, rather than axis duplication. Morpholino oligonucleotide-mediated reduction in ashwin expression leads to lethality or abnormalities in gastrulation, as well as significant apoptosis in midgastrula embryos. Apoptosis is also observed in midgastrula embryos overexpressing ashwin. Coexpression of ashwin with the bone morphogenetic protein-4 antagonist noggin has a synergistic effect on neural-specific gene expression in isolated animal cap ectoderm. Ashwin has no previously characterized domains, although two nuclear localization signals can be identified. Orthologues have been identified in the human, mouse, chicken, and pufferfish genomes. Our results suggest that ashwin regulates cell survival and anteroposterior patterning.

Amino Acid Sequence↗
PubMed2003

Functional expression of human heme oxygenase-1 gene in renal structure of spontaneously hypertensive rats.

Heme oxygenase (HO), by catabolizing heme to bile pigments, regulates the levels and activity of cellular hemoprotein and HO activity. We examined the effect of delivery of the human HO-1 gene on cellular heme in renal tissue using a retroviral vector. We used a single intracardiac injection of the concentrated infectious viral particles in 5-day-old spontaneously hypertensive rats; 25 were transduced with empty vector and 25 were transduced with the human HO-1 gene. Functional expression of human and rat HO-1 was measured after 2 and 4 weeks. Reverse transcription polymerase chain reaction showed that human HO-1 mRNA was expressed as early as 2 weeks, with the highest levels in the kidney. Western blot analysis showed distribution of human HO-1 protein in rat kidney structures, predominantly in the thick ascending limb of the loop of Henle as well as in proximal tubules and preglomerular arterioles. These areas also demonstrated higher HO activity as measured by increased conversion of heme to bilirubin and carbon monoxide. Functional expression of the human HO-1 gene was associated with a decrease in blood pressure in 4- and 8-week-old spontaneously hypertensive rats. Compared with nontransduced rats, human HO-1 gene overexpression in transduced rats was associated with a 35% decrease in urinary 20-hydroxyeicosatetraenoic acid, a potent vasoconstrictor and an inhibitor of tubular Na(+) transport, which may be related to the decrease in blood pressure.

Animals↗
PubMed2026

Metagenomic insights into biogeochemical functional potential and resistome dynamics of PM2.5 microbial communities.

Atmospheric particulate matter harbors diverse microorganisms, yet their functional potential in biogeochemical cycling and the associated risks of resistome remain poorly understood. Here, we performed metagenomic sequencing on PM2.5 samples collected across four months to unravel the microbial genetic repertoire involved in methane, nitrogen, phosphorus, and sulfur cycling, as well as the resistome, and pathogen composition. A broad range of functional genes was detected for each biogeochemical cycle, with more than 65% of gene subtypes shared across all months, indicating conserved functional signatures. In contrast, more than 80% of the resistome showed temporal variation in abundance, with the lowest richness observed in March. Temporal shifts were also observed in resistome composition, with several resistance determinants reaching higher abundances in April and May. Network analysis indicated frequent co-occurrence among several pathogenic and opportunistic taxa. Contig-based profiling identified 51 potential pathogenic taxa, including 32 human- or animal-associated taxa. In addition, both PM10 and PM2.5 concentrations were associated with pathogen abundance and functional gene richness (e.g., antibiotic resistance genes and virulence factors). Together, this metagenomic survey suggests contrasting temporal patterns between conserved biogeochemical functional potential and more variable resistome-related traits in PM2.5 microbial communities. While constrained by limited temporal coverage and sample size, this study provides preliminary insights into the ecological and potential public health relevance of airborne microbial communities in urban environments.

Particulate Matter↗