S1 Nuclease Analysis Research Articles

A novel KPC-113 variant conferring carbapenem and ceftazidime-avibactam resistance in a multidrug-resistant Pseudomonas aeruginosa isolate

ObjectivesThis study aimed to characterize a novel KPC-113 variant from a clinical Pseudomonas aeruginosa isolate R20-14. MethodsGenomic DNA of R20-14 was subjected to Illumina and Oxford Nanopore sequencing. The horizontal transmission of plasmid was evaluated with conjugation experiments. Minimum inhibitory concentrations of bacterial strains were obtained using broth microdilution methods. KPC-113 detectability of different carbapenemase detection methods was tested. The kinetic parameters of KPC-113 were compared with those of KPC-2 by a spectrophotometer. Structure modelling and molecular docking of KPC-2 and KPC-113 were performed using Schrödinger. ResultsR20-14, a sequence type 3903 multidrug-resistant strain, was resistant to carbapenems and ceftazidime-avibactam (CZA) concurrently. S1-nuclease pulsed-field gel electrophoresis and genomic analysis revealed a blaKPC-113–carrying plasmid pR20-14, which resembled the previously reported type I KPC-encoding P. aeruginosa plasmids and exhibited a high conjugation frequency. KPC-113, with a glycine residue insertion between Ambler positions 266 and 267 in KPC-2, conferred both carbapenem and CZA resistance in DH5α and PAO1 transformants. Diagnostic tests showed that KPC-113 acted in a similar manner to KPC-2. Compared with KPC-2, KPC-113 presented reduced catalytic ability to carbapenems and ceftazidime, meanwhile responding poorly to avibactam inhibition. Modelling structure revealed that KPC-113 possibly had a more flattened binding pocket than KPC-2, leading to the change of ligand binding modes. ConclusionsKPC-113 is a novel KPC variant mediating both CZA resistance and carbapenem resistance. It is of great concern that blaKPC-113 could transfer horizontally with great efficiency and inactivate carbapenems and CZA simultaneously. Great efforts should be made to prevent its spread in clinical settings.

Prevalence of qnrS-positive Escherichia coli from chicken in Thailand and possible co-selection of isolates with plasmids carrying qnrS and trimethoprim-resistance genes under farm use of trimethoprim

One hundred and twenty chicken samples from feces (n = 80), the carcass surface at slaughter at 2 meat chicken farms (n = 20), and retail chicken meat from 5 markets (n = 20) collected during 2018 and 2019 were examined for the prevalence of plasmid-mediated quinolone resistance (PMQR) in Escherichia coli. We detected qnrS-positive E. coli in a total of 74 samples from feces (n = 59), the carcass surface (n = 7), and retail meat (n = 8). These 74 qnrS-positive isolates were tested for antimicrobial susceptibility to determine the minimum inhibitory concentrations (MICs) of certain antimicrobials and genetically characterized. Ampicillin-resistance accounted for 71 of the 74 isolates (96%), followed by resistance to oxytetracycline (57/74; 77%), enrofloxacin (ERFX) (56/74; 76%), sulfisoxazole (SUL) (56/74; 76%), trimethoprim (TMP) (49/74; 66%), and dihydrostreptomycin (48/74; 65%). All farm-borne SUL- and TMP-resistant isolates except one were obtained from samples from farm A where a combination of sulfadiazine and TMP was administered to the chickens. Concentrations of ERFX at which 50 and 90% of isolates were inhibited were 2 μg/mL and 32 μg/mL, respectively. Diverse pulsed-field gel electrophoresis (PFGE) patterns of XbaI-digested genomic DNA were observed in the qnrS-positive isolates from fecal samples. Several isolates from feces and the carcass surface had identical XbaI-digested PFGE patterns. S1-nuclease PFGE and Southern blot analysis demonstrated that 7 of 11 dfrA13-positive fecal isolates carried both the qnrS and dfrA13 genes on the same plasmid, and 2 of 3 dfrA1-positive isolates similarly carried both qnrS and dfrA1 on the same plasmid, although the PFGE patterns of XbaI-digested genomic DNA of the isolates were different. These results suggest that the qnrS gene is prevalent in chicken farms via horizontal transfer of plasmids and may partly be co-selected under the use of TMP.

Biofilms in hospital effluents as a potential crossroads for carbapenemase-encoding strains

Bacterial resistance to carbapenem, which is mainly due to the successful dissemination of carbapenemase-encoding genes, has become a major health problem. Few studies have aimed to characterize the level of resistance in the environment, notably in hospital wastewater, which is a likely hotspot for exchange of antibiotic resistance genes.In this work, we looked for the presence of imipenem-resistant bacteria and imipenem in the effluent of the teaching hospital of Clermont-Ferrand, France. Selective growth of bacteria from 14-day old biofilms formed in the pipe sewer showed that 22.1% of the isolates were imipenem-resistant and identified as Aeromonas (n = 23), Pseudomonas (n = 10), Stenotrophomonas (n = 4) and Acinetobacter (n = 1). Fifteen of these strains harbored acquired carbapenemase-encoding genes blaVIM (n = 11), blaOXA-48 (n = 2), blaGES (n = 1), blaNDM (n = 1). All isolates also harbored associated resistances to aminoglycosides, fluoroquinolones and/or tetracyclin. S1-nuclease pulsed-field gel electrophoresis analysis of eight selected isolates showed that four of them harbored one to two plasmids of molecular weight between 48.5 Kb and 194 Kb. In vitro transformation assays evidenced the presence of blaVIM and blaNDM on plasmids with the blaVIM harboring 80 Kb plasmid having conjugative capacity.The predicted environmental concentration of imipenem in the hospital effluent was 3.16 μg/L, suggesting that biofilm bacteria are subjected to sub-MICs of imipenem within the effluent. However, no imipenem molecule was detected in the hospital effluent, probably owing to its instability: in vitro assays indicated that imipenem's biological activity was no longer detectable after 45 h of storage. However, the predictive value of the hazard quotient relative to the development of resistance was >1.0 (HQr = 28.9 ± 1.9), which indicates a possible risk.The presence of carbapenemase-encoding genes in hospital effluent biofilm strains and their ability to transfer are therefore a potential hazard that should not be neglected and points to the need for monitoring antibiotic resistance in hospital wastewater.

Antimicrobial resistance profiles and molecular characterization of Escherichia coli strains isolated from healthy adults in Ho Chi Minh City, Vietnam.

In this study, we attempted to isolate Escherichia coli from healthy adults in Ho Chi Minh City, Vietnam, and characterized its antimicrobial resistance profile, extended-spectrum β-lactamase (ESBL) genotype, phylogenetic grouping and virulence gene profile. A total of 103 E. coli isolates were obtained, and most of them were antimicrobial resistant such to streptomycin (80.6%), tetracycline (67.0%), ampicillin (65.0%), sulfamethoxsazole/trimethoprim (48.5%), nalidixic acid (43.7%), chloramphenicol (34.0%), cefotaxime (15.5%), ciprofloxacin (15.5%), kanamycin (12.6%), ceftazidime (10.7%), fosfomycin (4.9%) and gentamicin (2.9%). However, all these E. coli strains were susceptible to imipenem. Surprisingly, of 103 strains, 74 (71.8%) and 43 (41.7%) strains showed resistance to more than 3 and 5 classes of antimicrobials, respectively. Furthermore, 10 E. coli strains were ESBL-producers and positive for blaCTX-M genes (7 for blaCTX-M-9 and 3 for blaCTX-M-1), while five were additionally positive for blaTEM genes. S1-nuclease pulsed-field gel electrophoresis analysis revealed that 7 and 3 strains of E. coli carry blaCTX-M genes on their large plasmid and chromosome, respectively. Phylogenetic analysis exhibited that majority of the E. coli strains was grouped into A (44.7%), followed by B1 (23.3%), B2 (18.4%) and D (13.6%). Virulence genes associated with diarrheagenic E. coli, such as astA, EAF, eaeA, elt and eagg were also detected in ESBL-producing E. coli as well as antimicrobial resistant strains. These data suggest that commensal E. coli of healthy human could be a reservoir for antimicrobial resistance determinants and some of them might be harmful to human.

PCR-based plasmid typing inEnterococcus faeciumstrains reveals widely distributed pRE25-, pRUM-, pIP501- and pHTβ-related replicons associated with glycopeptide resistance and stabilizing toxin–antitoxin systems

A PCR-based typing scheme was applied to identify plasmids in an epidemiologically and geographically diverse strain collection of Enterococcus faecium (n=93). Replicon types of pRE25 (n=56), pRUM (n=41), pIP501 (n=17) and pHTbeta (n=14) were observed in 83% of the strains, while pS86, pCF10, pAM373, pMBB1 or pEF418 were not detected. Furthermore, 61% of the strains contained the axe-txe (n=42) or/and the omega-epsilon-zeta (n=18) plasmid stabilization loci. Sequence analyses divided the omega-epsilon-zeta operon into two distinct phylogenetic groups. The present typing scheme accounted for about 60% of the total number of plasmids detected by S1 nuclease analyses, which revealed zero to seven plasmids (10 kb to >200 kb) per isolate. Interestingly, strains belonging to the clinically important clonal complex 17 (CC17) yielded a significantly higher number of plasmids (3.1) and pRUM replicons (74%) than non-CC17 strains (2.2% and 35%, respectively). A prevalent genetic linkage between the pRUM-replicon type and axe-txe was demonstrated by cohybridization analyses. The vanA resistance determinant was associated with all four replicon types, but we also confirmed the genetic linkage of vanA to unknown transferable replicons. PCR-based replicon typing, linked to the detection of other important plasmid-encoded traits, seems to be a feasible tool for tracing disseminating resistance plasmids stably maintained in various environments.

Chromosomal Integration of the Extended-Spectrum β-Lactamase Gene bla CTX-M-15 in Salmonella enterica Serotype Concord Isolates from Internationally Adopted Children

We report the emergence of Salmonella enterica isolates of serotype Concord (and its monophasic variant 6,7:l,v:-) producing the extended-spectrum beta-lactamases (ESBLs) SHV-12 and CTX-M-15 in France and Norway between 2001 and 2006 (43 in France and 26 in Norway). The majority of these isolates were from adopted children from Ethiopia, most of whom were healthy carriers. Several symptomatic secondary cases were found in the adoptive families and health care facilities in France. Serotype Concord isolates collected before 2003 produced SHV-12 encoded on a 340-kb conjugative plasmid of replicon IncI1. Isolates collected after 2003 produced CTX-M-15. We detected two conjugative plasmids carrying bla(CTX-M-15). One plasmid, approximately 300 kb in size, was positive for the IncHI2 replicon and the plasmid-mediated quinolone resistance gene qnrA1. The other plasmid, from one of the earliest CTX-M-15-producing isolates collected, was a fusion plasmid with IncY and IncA/C(2) replicons and was 200 kb in size. However, we showed, using Southern hybridization of I-CeuI-digested chromosomal DNA and S1 nuclease analysis of plasmid DNA, that most isolates had a bla(CTX-M-15) gene located on chromosomal DNA. Analysis of the flanking regions of the chromosomally located bla(CTX-M-15) gene by cloning revealed an ISEcp1 truncated by an intact IS26 upstream from the bla(CTX-M-15) gene and a truncated orf477 gene downstream from bla(CTX-M-15). We found regions beyond the IS26 and the orf477 genes that were derived from IncA/C(2) plasmids, suggesting the chromosomal integration of part of the bla(CTX-M-15)-carrying IncY and IncA/C(2) fusion plasmid from early CTX-M-15-producing isolates.

Characterization of a rolling-circle replication plasmid from Thermus aquaticus NTU103

The thermophilic bacterium Thermus aquaticus NTU103 harbors a 1965-bp plasmid, pTA103. Sequencing analysis revealed that pTA103 contains two open reading frames. One of the open reading frames ( orf2) shares no significant homology with protein in the data bank. The other one has 50% similarity and 34% identity with RepA-like protein of pRm1132f, which is a rolling-circle replication (RCR) plasmid isolated from Sinorhizobium meliloti. S1 nuclease analysis demonstrated that pTA103 contains a single-stranded intermediate, confirming that pTA103 replicates via RCR mechanism. Sequence data also revealed putative double-stranded origin and single-stranded origin sites, indicating the importance of these cis elements in pTA103 replication.

Sequence Analysis of Two Cryptic Plasmids from Bifidobacterium longum DJO10A and Construction of a Shuttle Cloning Vector

Bifidobacterium longum DJO10A is a recent human isolate with probiotic characteristics and contains two plasmids, designated pDOJH10L and pDOJH10S. The complete sequences of both these plasmids have now been determined and consist of two circular DNA molecules of 10,073 and 3,661 bp, with G+C contents of 62.2% and 66.2%, respectively. Plasmid pDOJH10L is a cointegrate plasmid consisting of DNA regions exhibiting very high sequence identity to two other B. longum plasmids, pNAC2 (98%) and pKJ50 (96%), together with another region. Interestingly, the rolling circular replication (RCR) regions of both the pNAC2- and pKJ50-like plasmids were disrupted during the recombination event leading to a further recombination event to acquire a functional replicon. This consists of a new fused rep gene and an RCR-type ori consisting of a conserved DnaA box in an AT-rich region followed by four contiguous repeated sequences consistent with an iteron structure and an inverted repeat. The smaller pDOJH10S had no sequence similarity to any other characterized plasmid from bifidobacteria. In addition, it did not contain any features consistent with RCR, which is the replication mechanism proposed for all the bifidobacteria plasmids characterized to date. It did exhibit sequence similarity with several theta replication-related replication proteins from other gram-positive, high-G+C bacteria, with the closest match from a Rhodococcus rhodochrous plasmid, suggesting a theta mechanism of replication. S1 nuclease analysis of both plasmids in B. longum DJO10A revealed single-strand DNA intermediates for pDOJH10L, which is consistent for RCR, but none were detected for pDOJH10S. As the G+C content of pDOJH10S is similar to that of Rhodococcus rhodochrous (67%) and significantly higher than that of B. longum (60.1%), it may have been acquired through horizontal gene transfer from a Rhodococcus species, as both genera are members of the Actinomycetes and are intestinal inhabitants. An Escherichia coli-B. longum shuttle cloning vector was constructed from pDOJH10S and the E. coli ori region of p15A, a lacZ gene with a multiple cloning site of pUC18, and a chloramphenicol resistance gene (CAT) of pCI372 and was transformed successfully into E. coli and B. longum. It could not be introduced into lactic acid bacteria (Lactococcus and Lactobacillus), showing it was not very promiscuous. It was stably maintained in B. longum in the absence of antibiotic pressure for 92 generations, which is consistent with the segregational stability of theta-replicating plasmids in gram-positive bacteria. This is the first cloning vector for bifidobacteria that does not utilize RCR and should be useful for the stable introduction of heterologous genes into these dominant inhabitants of the large intestine.

Control of MYEOV Protein Synthesis by Upstream Open Reading Frames

The myeov gene has been isolated by the tumorigenicity assay and is localized at chromosome 11q13, a frequent site for chromosomal rearrangements in various carcinomas and B-cell neoplasms. In addition, myeov is coamplified with cyclin D1 and overexpressed in carcinomas of various organs. The mechanisms of myeov regulation remain enigmatic. The 5'-untranslated region (5'-UTR) of the myeov gene is long, encompasses several upstream AUGs, and is predicted to fold in a strong secondary structure, suggesting that its translation might be regulated by an internal ribosomal entry site. Here we show that initial experiments using monocistronic and dicistronic reporter constructs supported this assumption. However, the application of in vitro transcription/translation assays, Northern blot analysis, and promoterless dicistronic constructs revealed promoter activity of the myeov 5'-UTR. DNA transfection of dicistronic DNA constructs, normal and mutated forms of myeov cDNA fragments cloned in a eukaryotic expression vector, and direct RNA transfection analysis revealed that upstream AUG triplets in the 5'-UTR of the myeov transcript abrogate translation. Alternative splicing mechanisms in specific cell types and/or developmental stage may evade this translation control. Control experiments suggest that the 5'-UTR from encephalomyocarditis virus, when inserted at the midpoint of a dicistronic vector, is also able to function as a cryptic promoter.

The gonadotropin-regulated long-chain acyl CoA synthetase gene: A novel downstream Sp1/Sp3 binding element critical for transcriptional promoter activity

The 79 kD gonadotropin-regulated testicular long chain acyl-CoA synthetase gene (GR-LACS) is a hormone-regulated member of the acyl-CoA synthetase family that is expressed abundantly in Leydig cells and to a lesser extent in germinal cells of the adult testis. GR-LACS possesses an ATP/AMP binding domain and the fatty acyl-CoA synthetase (FACS) signature motif. To gain insights into the transcriptional regulation of GR-LACS in gonadal cells, we determined the genomic organization of the gene, including the upstream flanking sequences. The mouse GR-LACS gene spans over at least 45 kb and the coding region is encoded by exons 1–14. All exon–intron junction sites correspond to the consensus splice sequence GT-AG. Exon 7 and 11 comprise the conserved ATP/AMP binding domain and the FACS signature motif, respectively. Primer extension and S1 nuclease analyses demonstrated four transcriptional start sites located at − 266 / − 216 bp 5′ to the ATG codon. The minimal promoter domain resides within − 254 / − 217 bp 5′ to ATG codon, and upstream sequences to − 404 bp (− 1035 / − 405 bp) contribute to the inhibition of transcription in the expressing mouse Leydig tumor cells. Removal of − 217 / − 1 bp, containing a 23 nt GC rich sequence (− 112 / − 90) with an Sp1/Sp3 binding element, within the 1st exon of this TATA-less promoter, significantly reduced GR-LACS gene transcription. Transcriptional activity was abolished by a 2 nt mutation of this element. Thus, functional analyses of this promoter domain indicate that transcription of GR-LACS gene requires an Sp1/Sp3 binding element downstream of the transcriptional start sites which is essential for basal promoter activity.

The AUUCU Repeats Responsible for Spinocerebellar Ataxia Type 10 Form Unusual RNA Hairpins

Spinocerebellar ataxia type 10 is an autosomal dominant disorder caused by expansion of a pentameric repeat tract (ATTCT.AGAAT)(n) in intron 9 of the gene that encodes ataxin-10. We have analyzed duplex DNA containing the repeat, the individual DNA strands, and the RNA that would be generated by transcription of the repeat. Circular dichroism and UV absorbance measurements suggest that the previously reported tendency of the repeat to unpair when supercoiled is probably related simply to GC content rather than reflecting any unusual property of the duplex. DNA containing d(ATTCT)9 forms a folded structure at relatively low temperatures, whereas the antisense strand, d(AGAAT)9, does not form a structure even at 0 degrees C. In contrast r(AUUCU)9 forms a folded structure under physiologically reasonable conditions. S1 nuclease analysis reveals a single region of hypersensitivity in the middle of the repeat tract, whereas V1 digestion is consistent with a hydrogen bonded or well stacked structure. CD spectroscopy shows that the structure is unimolecular and hydrogen bonded and has a significant amount of A-form helix. NMR spectroscopy demonstrates that these hydrogen bonds comprise an equal number of A.U and U.U base pairs. Our data thus suggest that the repeat forms an unusual RNA hairpin. Thus the ability to form an RNA hairpin seems to be a common property of those Repeat Expansion Diseases that are not recessively inherited and are caused by repeats that are transcribed but not translated.

Genome-wide Analysis of Iron-dependent Growth Reveals a Novel Yeast Gene Required for Vacuolar Acidification

We conducted a genome-wide screen in the budding yeast Saccharomyces cerevisiae of 4,792 homozygous diploid deletions to identify genes that function in iron metabolism. Strains unable to grow on iron-restricted medium contained deletions of genes that encode the structural components of the high affinity iron transport system (FET3, FTR1), the iron-sensing transcription factor AFT1 or genes required for the assembly of the transport system. We also identified genes that were not previously known to play a role in iron metabolism. Deletion of the gene CWH36 resulted in a severe growth defect on iron-limited medium, as well as increased sensitivity to Congo red and calcofluor white. Iron transport studies demonstrated that Deltacwh36 cells have an inability to copper load apoFet3p. Furthermore, Deltacwh36 cells demonstrated additional phenotypes including distorted vacuole morphology and altered kinetics of FM4-64 trafficking. We show that Deltacwh36 cells have a defect in vacuolar acidification through the use of the pH-sensitive dye LysoSensor Green DND-189. In Deltacwh36 cells, the vacuolar H+-ATPase is not assembled and there are reduced levels of at least one subunit of the V0 complex. The open reading frame responsible for the Deltacwh36 phenotypes is YCL005W-A. This gene contains two introns, has homologues in other Saccharomyces strains, and shows weak homology to a component of the vacuolar H+-ATPase found in organisms as diverse as insect and cow.

Genomic organization and promoter analysis of the Dnmt3b gene

The Dnmt3b gene encodes a de novo DNA methyltransferase that is essential for normal mouse development. It is highly expressed in early embryos and embryonic stem (ES) cells but downregulated in most adult somatic tissues. To gain insight into the regulation of Dnmt3b, we have isolated a mouse genomic bacterial artificial chromosome clone that contains the Dnmt3b gene. Complete sequence analysis of the clone demonstrated that Dnmt3b consists of at least 24 exons and spans 38 kilobases. S1 nuclease analysis identified two adjacent transcriptional start sites located downstream of a unique TATA-like element in a CpG island. There was an unknown gene which we named mU 3 17 kb upstream of the Dnmt3b locus, and it was transcribed ubiquitously and in the opposite direction of Dnmt3b. Transfection analysis revealed that the minimal promoter region containing an Sp1 site was active even in somatic cells, and that there were several repressor elements within 7.9 kb upstream of Dnmt3b downregulated this gene specifically in somatic cells but not in ES cells. These findings provide a basis for future detailed studies of the mechanisms controlling Dnmt3b expression.

Generation and characterization of two transgenic mouse lines expressing human ApoE2 in neurons and glial cells.

Apolipoprotein E (apoE) isoforms are key determinants of susceptibility to late-onset Alzheimer's disease (AD). The epsilon 4 and epsilon 2 alleles have been associated with increased and decreased risk for AD, respectively. We have generated and characterized transgenic mice in which the human apoE2 gene is expressed under the control of the platelet-derived growth factor B-chain (PDGF-B) promoter, or the transferrin (TF) promoter. S1 nuclease analysis and immunoblotting showed that the PDGF-B apoE2 mice express apoE2 exclusively in the brain whereas the TF apoE2 mice express apoE2 in the liver and in the brain. In the TF apoE2 mouse line, apoE2 is also detected in the plasma. The PDGF-B apoE2 and the TF apoE2 transgenic mice were bred back to apoE(-)(/)(-) background. Immunohistochemical analysis showed that the PDGF apoE2 x apoE(-)(/)(-) and the TF apoE2 x apoE(-)(/)(-) mice express human apoE2 within the neocortex in hippocampal neurons and glial cells, respectively. ApoE(-)(/)(-) mice have been shown to develop age-dependent loss of synaptophysin. Immunoblotting of mouse brain extracts and immunohistochemical analysis of brain sections showed that apoE expression in both apoE2 x apoE(-)(/)(-) transgenic lines was associated with significant recovery of brain synaptophysin levels as compared to the levels of apoE(-)(/)(-) littermates of the same age. These apoE2-expressing mice, when bred back on amyloid precursor protein (APP) transgenic mice or other mouse lines featuring alterations in lipoprotein metabolism, may provide new mouse models for elucidating the role of apoE2 in lipid homeostasis in the brain and in the pathogenesis of AD.

Open reading frame UL26 of human cytomegalovirus encodes a novel tegument protein that contains a strong transcriptional activation domain.

A selection strategy, the activator trap, was used in order to identify genes of human cytomegalovirus (HCMV) that encode strong transcriptional activation domains in mammalian cells. This approach is based on the isolation of activation domains from a GAL4 fusion library by means of selective plasmid replication, which is mediated in transfected cells by a GAL4-inducible T antigen gene. With this screening strategy, we were able to isolate two types of plasmids encoding transactivating fusion proteins from a library of random HCMV DNA inserts. One plasmid contained the exon 3 of the HCMV IE-1/2 gene region, which has previously been identified as a strong transcriptional activation domain. In the second type of plasmid, the open reading frame (ORF) UL26 of HCMV was fused to the GAL4 DNA-binding domain. By quantitative RNA mapping using S1 nuclease analysis, we were able to classify UL26 as a strong enhancer-type activation domain with no apparent homology to characterized transcriptional activators. Western blot analysis with a specific polyclonal antibody raised against a prokaryotic UL26 fusion protein revealed that two protein isoforms of 21 and 27 kDa are derived from the UL26 ORF in both infected and transfected cells. Both protein isoforms, which arise via alternative usage of two in-frame translational start codons, showed a nuclear localization and could be detected as early as 6 h after infection of primary human fibroblasts. By performing Western blot analysis with purified virions combined with fractionation experiments, we provide evidence that pUL26 is a novel tegument protein of HCMV that is imported during viral infection. Furthermore, we observed transactivation of the HCMV major immediate-early enhancer-promoter by pUL26, whereas several early and late promoters were not affected. Our data suggest that pUL26 is a novel tegument protein of HCMV with a strong transcriptional activation domain that could play an important role during initiation of the viral replicative cycle.

A promoter activity in Saccharomyces cerevisiae of the 3'-noncoding region of the basidiomycetous mushroom gene.

The 3'-noncoding region of the priB gene derived from the basidiomycete Lentinus edodes was found to contain one GC box-like sequence, two CAAT boxes, two TATA box-like sequences and two pyrimidine-rich stretches (CT-motifs). It also contained a 16-bp sequence similar to the consensus sequence for PRIB protein binding and a short (61 bp) intron. Single-strand-specific S1 nuclease analysis of plasmid pBR322 DNA containing the 3'-noncoding region propagated in Escherichia coli revealed that this region forms an unusual, extended open structure within/around the downstream pyrimidine/purine (CT/AG)-biased sequence. To examine the promoter activity of the priB 3'-noncoding region in Saccharomyces cerevisiae, in which an autonomously replicating plasmid vector is available, we constructed two plasmids, YEp3'NCR-lacZ and YEp3'UTR-lacZ. The former contains the priB 3'-noncoding region and the reporter E. coli beta-galactosidase gene while the latter contains the priB 3'-noncoding region lacking the intron and the reporter gene. The yeast transformants obtained through introductions of these plasmids showed beta-galactosidase activity and the activity conferred by YEp3'NCR-lacZ was about 50% of that conferred by YEp3'UTR-lacZ. The primer extension analysis showed that transcription of the reporter gene on both plasmids starts at three alternative sites all of which are located within the downstream CT-motif, suggesting a role for the unusual structure of the CT/AG-biased sequence in the initiation of transcription.

A nonsense mutation is responsible for the RNA-negative phenotype in human citrullinaemia

Citrullinaemia is an inborn error of metabolism resulting from a deficiency of argininosuccinate synthetase. Previous studies of RNA of argininosuccinate synthetase of citrullinaemia patients using S1 nuclease analysis have identified a class of so-called RNA-negative alleles in which no stable mRNA can be detected. To investigate the nature of mutation responsible for such a phenotype, a compound heterozygous citrullinaemia carrying an RNA-negative allele and an allele with a 3' splice site mutation in intron 6 (IVS6-2A>G) was analysed. Using sequences of a DNA polymorphism and the IVS6-2A>G mutation as markers, approximately equal amounts of pre-mRNAs from allelic genes were detected suggesting that RNA-negative phenotype could not be the result of defect in transcription initiation. A C-to-T transition converting the CGA arginine codon at residue 279 to a TGA termination codon (R279X) was identified by cDNA sequencing. No accumulation of partially spliced pre-mRNAs containing introns immediately upstream and downstream of the nonsense mutation was observed. In addition, no mRNA species of abnormal size was detected when cDNA from the RNA-negative allele was analysed. Hence, there is no indication of nonsense-associated altered splicing (NAS). The most likely event responsible for the RNA-negative phenotype appears to be nonsense-mediated mRNA decay (NMD).

Differential Regulation of the Aldehyde Dehydrogenase 1 Gene in Embryonic Chick Retina and Liver

Aldehyde dehydrogenase (ALDH1) is highly expressed in the dorsal cells of the undifferentiated retina, where it has been proposed to play a role in the formation of a retinoic acid gradient along the ventrodorsal axis. In contrast to the retina, ALDH1 levels increase with differentiation in the liver and remain elevated in the adult tissue. To understand the molecular basis for differential expression of ALDH1 during development, we characterized the ALDH1 transcripts expressed in chick retina and liver. By sequencing, primer extension, and S1 nuclease analysis, we show that retina ALDH1 mRNA has an additional 300 nucleotides of 5'-untranslated sequence resulting from the transcription of two 5' noncoding exons. There is a 24-29-kilobase pair (kb) gap between exons 1 and 2 and a 290-base pair gap between exons 2 and 3. Exon 3, which contains the ALDH1 start codon, represents the first exon of the liver transcript. Using a reporter gene assay, we have identified tissue-specific regulatory elements that govern ALDH1 expression in primary retina and liver cultures. Constructs with >1.6 kb of DNA flanking the 5'-end of exon 1 showed elevated activity in retinal cultures but only basal activity in liver cultures. In contrast, constructs with <1 kb of 5'-flanking DNA were active in both retina and liver cultures. Our results suggest that an important mechanism for the control of ALDH1 transcriptional activity is through the presence of inhibitory elements located 0.7-1.6 kb upstream of the ALDH1 gene. DNase I footprint analysis reveal four sites of protein-DNA interaction within this region, one of which is specific to the liver and corresponds to a NF-kappaB/Rel binding site.

Molecular Characterizations of the Equine Herpesvirus 1 ETIF Promoter Region and Translation Initiation Site

The equine herpesvirus 1 (EHV-1) homolog of the herpes simplex virus type 1 (HSV-1) tegument phosphoprotein, αTIF (Vmw65; VP16), was identified previously as the product of open reading frame 12 (ORF12), was shown to trans-activate immediate-early (IE) gene promoters, and was described as a 60-kDa virion component designated ETIF. However, the ETIF promoter region and transcription initiation site were not identified. The poly(A) signal of the gene 11 (UL49 homolog) lies just upstream of the first ETIF translation initiation codon, indicating that the first ATG may not be used for initiating ETIF translation. Another in-frame translation initiation codon (ATG2) is located 88 bp downstream of the first ETIF initiation codon (ATG1). Western blot analysis showed that the expressed ETIF protein migrated in SDS–PAGE with an apparent molecular mass of approximately 56 kDa, the same molecular weight identified in SDS–PAGE analysis of the KyD EHV-1 virion preparations. The ETIF expression vector pCETIF, which contains ATG2, trans-activated the IE promoter more efficiently than the pC12 containing both ATG1 and ATG2. S1 nuclease analyses mapped the 5′ initiation site of the 1.4-kb transcript approximately 17 to 21 nt downstream of the ATG1. The nucleotide sequence upstream of the ATG1 did not have any promoter activity, while the nucleotide sequence upstream of the ATG2 had promoter activity. In transient transfection assays, the pETIFM2 vector, which was mutated in the ATG2, did not trans-activate the IE promoter; however, the pETIFM1 vector, which was mutated in the ATG1, trans-activated the IE promoter. These results demonstrated that the ATG2 of the ETIF ORF is the ETIF translation initiation codon. ETIF trans-activated only the IE promoter, not early (EICP0, EICP22, EICP27, and thymidine kinase) or late (IR5) promoters, confirming that EICP0, EICP22, and EICP27 are early genes.

The aprX-lipA operon of Pseudomonas fluorescens B52: a molecular analysis of metalloprotease and lipase production.

Extracellular protease and lipase production by psychrotrophic strains of Pseudomonas fluorescens is repressed by iron and regulated by temperature. The regulation of protease and lipase has been investigated in P. fluorescens B52. Whereas lipase production is increased below the optimum growth temperature ('low-temperature regulation'), protease production was relatively constant and only decreased above the optimum growth temperature. The genes encoding protease (aprX) and lipase (lipA) are encoded at opposite ends of a contiguous set of genes which also includes protease inhibitor, Type I secretion functions and two autotransporter proteins. Evidence is presented indicating that these genes constitute an operon, with a promoter adjacent to aprX which has been identified by S1 nuclease analysis. The regulation of aprX and lipA has been investigated at the RNA level and using lacZ fusion strains. Whereas the data are consistent with iron regulation at the transcriptional level, a lipA'-'lacZ fusion is not regulated by temperature, suggesting that temperature regulation is post-transcriptional or post-translational. The possibility of regulation at the level of mRNA decay is discussed.