Upstream Copy Research Articles

Genomic Characterization of International High-Risk Clone ST410 Escherichia coli Co-Harboring ESBL-Encoding Genes and blaNDM-5 on IncFIA/IncFIB/IncFII/IncQ1 Multireplicon Plasmid and Carrying a Chromosome-Borne blaCMY-2 from Egypt.

The accelerated dispersion of multidrug-resistant (MDR) Escherichia coli due to the production of extended-spectrum β-lactamases (ESBLs) or AmpC enzymes has been noted in Egypt, presenting a serious treatment challenge. In this study, we investigate the prevalence of ESBLs and AmpC enzymes among 48 E. coli isolates collected from patients with urinary tract infections admitted to a teaching hospital in Alexandria. Phenotypic and genotypic methods of detection are conducted. Isolates producing both enzymes are tested for the mobilization of their genes by a broth mating experiment. Whole genome sequencing (WGS) is performed for isolate EC13655. The results indicate that 80% of the isolates are MDR, among which 52% and 13% were ESBL and AmpC producers, respectively. Conjugation experiments fail to show the mobilization of blaCMY-2 in EC13655, which was chosen for WGS. In silico analysis reveals that the isolate belongs to a ST410-H24Rx high-risk clone. It coharbors the ESBL-encoding genes blaCTX-M-15, blaTEM-1, blaOXA-1 and blaNDM-5 on an IncFIA/IncFIB/IncFII/IncQ1 multireplicon plasmid. The chromosomal location of blaCMY-2 is detected with a flanking upstream copy of ISEcp1. This chromosomal integration of blaCMY-2 establishes the stable maintenance of the gene and thus, necessitates an imperative local surveillance to reduce further spread of such strains in different clinical settings.

A Model for Transposition of the Colistin Resistance Gene mcr-1 by ISApl1.

Analysis of mcr-1-containing sequences identified a common ∼2,607-bp DNA segment that in many cases is flanked on one or both ends by ISApl1 We present evidence that mcr-1 is mobilized by an ISApl1 composite transposon which has, in some cases, subsequently lost one or both copies of ISApl1 We also show that mcr-1 can be mobilized in some circumstances by a single upstream copy of ISApl1 in conjunction with the remnants of a downstream ISApl1.

Intron gain by tandem genomic duplication: a novel case in a potato gene encoding RNA-dependent RNA polymerase.

The origin and subsequent accumulation of spliceosomal introns are prominent events in the evolution of eukaryotic gene structure. However, the mechanisms underlying intron gain remain unclear because there are few proven cases of recently gained introns. In an RNA-dependent RNA polymerase (RdRp) gene, we found that a tandem duplication occurred after the divergence of potato and its wild relatives among other Solanum plants. The duplicated sequence crosses the intron-exon boundary of the first intron and the second exon. A new intron was detected at this duplicated region, and it includes a small previously exonic segment of the upstream copy of the duplicated sequence and the intronic segment of the downstream copy of the duplicated sequence. The donor site of this new intron was directly obtained from the small previously exonic segment. Most of the splicing signals were inherited directly from the parental intron/exon structure, including a putative branch site, the polypyrimidine tract, the 3′ splicing site, two putative exonic splicing enhancers, and the GC contents differed between the intron and exon. In the widely cited model of intron gain by tandem genomic duplication, the duplication of an AGGT-containing exonic segment provides the GT and AG splicing sites for the new intron. Our results illustrate that the tandem duplication model of intron gain should be diverse in terms of obtaining the proper splicing signals.

Determination of the human SULT1C3 mRNA sequence in LS180 cells

The human SULT1C subfamily consists of SULT1C2, SULT1C4, and one putative gene, SULT1C3, which has been identified by computational analysis of the human genome but has not been reported as expressed in any human tissue. An unusual feature of the predicted SULT1C3 gene is that it contains a duplication of the last two exons, with the upstream copies designated as exons 7a and 8a and the downstream copies designated as exons 7b and 8b. The NCBI reference sequence for SULT1C3 mRNA (NM_001008743) begins at the predicted translation start site, ends with exons 7b and 8b, and does not include exon 7a or 8a. We used primers corresponding to sequences in exons 6 and 7b to survey SULT1C3 expression in human tissues and amplified a largerthan‐expected RT‐PCR fragment from small and large intestinal mRNA as well as from LS180 human colorectal adenocarcinoma cells. The cloned fragment contained exons 7a and 8a in addition to the expected portion of exon 7b. We then used 5′‐and 3′‐RACE to determine the entire SULT1C3 mRNA sequence in LS180 cells. These analyses indicated that the SULT1C3 gene contains a non‐coding exon ~7.1 kb upstream of the first coding exon and that the major SULT1C3 transcript contains exons 7a and 8a rather than 7b and 8b. We conclude that SULT1C3 is expressed in human intestinal cells but that its 3′‐exons are not those indicated for the reference sequence. This work was supported by NIH grants HL050710 and ES005823.

Homologous Recombination Assay

Repair of double strand break by homologous recombination was examined using U2OS cells or RG37 cells harbouring specific substrate developed by Puget et al. (2005) and Dumay et al. (2006), respectively, to measure the repair of DNA double strand breaks by homologous recombination. The substrate is composed of two inactive copies of the GFP gene. The upstream copy is inactive due to the absence of promoter, the downstream copy present a promoter but is inactivated by the insertion of the sequence coding for the recognition site of the I-SceI enzyme. The substrate is stably expressed in cells after its insertion in the genome and present as a unique copy. The unique DNA double strand break is then induced by the expression of the I-SceI enzyme after cell transfection with a plasmid coding for the I-SceI enzyme., 使用由Puget等人开发的含有特异性底物的U2OS细胞或RG37细胞检查通过同源重组修复双链断裂。 （2005）和Dumay等人 （2006），分别通过同源重组来测量DNA双链断裂的修复。 底物由GFP基因的两个无活性拷贝组成。 上游拷贝由于不存在启动子而不活跃，下游拷贝存在启动子，但通过插入编码I-SceI酶的识别位点的序列而失活。 底物在插入基因组后稳定地表达在细胞中，作为唯一拷贝存在。 然后通过用编码I-SceI酶的质粒转染细胞后，通过I-SceI酶的表达来诱导独特的DNA双链断裂。

Genes Involved in the Degradation of Ether Fuels by Bacteria of the Mycobacterium/Rhodococcus Group

A cluster of genes encoding a cytochrome P-450 monooxygenase system involved in the utilisation of ethyl tert-butyl ether (ETBE) was cloned in Rhodococcus ruber IFP 2001. This cluster includes ethR, a putative regulator gene of the araC/xylS family; ethA, encoding a ferredoxin reductase; ethB, encoding a cytochrome P-450, ethC, encoding a ferredoxin; and ethD, which is required for the function of the monooxygenase system, but whose exact role is unknown. The ethRABCD cluster is flanked on either side by two identical copies of a class II transposon, which explains that it is lost at high frequency by homologous recombination when the strain is grown under non selective conditions. Two other, highly conserved clusters of eth genes were detected in the ETBE-utilizing strains Rhodococcus zopfii IFP 2005 and Mycobacterium sp. IFP 2009. In all cases, the eth locus is inserted in a different genomic context, suggesting that it may be transferred horizontally between different species and inserted at different sites in the genome. In addition, in R. zopfii IFP 2005, the downstream copy of the transposon carries a 117-bp (base pairs) deletion; in Mycobacterium sp. IFP 2009, the upstream copy is absent and the downstream copy is inserted 2771 bp closer to the ethRABCD cluster.

Evolution of the human immunodeficiency virus type 1 long terminal repeat promoter by conversion of an NF-kappaB enhancer element into a GABP binding site.

Human immunodeficiency virus type 1 (HIV-1) transcription is regulated by the viral Tat protein and cellular factors, of which the concentration and activity may depend on the cell type. Viral long terminal repeat (LTR) promoter sequences are therefore optimized to suit the specific nuclear environment of the target host cell. In long-term cultures of a Tat-defective, poorly replicating HIV-1 mutant, we selected for a faster-replicating virus with a 1-nucleotide deletion in the upstream copy of two highly conserved NF-kappaB binding sites. The variant enhancer sequence demonstrated a severe loss of NF-kappaB binding in protein binding assays. Interestingly, we observed a new binding activity that is specific for the variant NF-kappaB sequence and is present in the nuclear extract of unstimulated cells that lack NF-kappaB. These results suggest that inactivation of the NF-kappaB site coincides with binding of another transcription factor. Fine mapping of the sequence requirements for binding of this factor revealed a core sequence similar to that of Ets binding sites, and supershift assays with antibodies demonstrated the involvement of the GABP transcription factor. Transient transfection experiments with LTR-chloramphenicol acetyltransferase constructs indicated that the variant LTR promoter is specifically inhibited by GABP in the absence of Tat, but this promoter was dramatically more responsive to Tat than the wild-type LTR. Introduction of this GABP site into the LAI virus yielded a specific gain of fitness in SupT1 cells, which contain little NF-kappaB protein. These results suggest that GABP potentiates Tat-mediated activation of LTR transcription and viral replication in some cell types. Conversion of an NF-kappaB into a GABP binding site is likely to have occurred also during the worldwide spread of HIV-1, as we noticed the same LTR modification in subtype E isolates from Thailand. This typical LTR promoter configuration may provide these viruses with unique biological properties.

Transcriptional Repression by v-Ski and c-Ski Mediated by a Specific DNA Binding Site

The Ski oncoprotein has been shown to bind DNA and activate transcription in conjunction with other cellular factors. Because tumor cells or myogenic cells were used for those studies, it is not clear that those activities of Ski are related to its transforming ability. In this study, we use a nuclear extract of c-ski-transformed cells to identify a specific DNA binding site for Ski with the consensus sequence GTCTAGAC. We demonstrate that both c-Ski and v-Ski in nuclear extracts are components of complexes that bind specifically to this site. By evaluating the features of the sequence that are critical for binding, we show that binding is cooperative. Although Ski cannot bind to this sequence on its own, we use cross-linking with ultraviolet light to show that Ski binds to this site along with several unidentified cellular proteins. Furthermore, we find that Ski represses transcription either through upstream copies of this element or when brought to the promoter by a heterologous DNA binding domain. This is the first demonstration that Ski acts as a repressor rather than an activator and could provide new insights into regulation of gene expression by Ski.

Long-distance effect of downstream transcription on activity of the supercoiling-sensitive leu-500 promoter in a topA mutant of Salmonella typhimurium

Expression of the lacZ gene from the supercoiling-sensitive leu-500 promoter on a plasmid in topA mutant cells was stimulated by activating a divergently oriented Tac promoter, 400 bp upstream from leu-500. The stimulation was approximately threefold regardless of whether the Tac promoter drove the expression of the tet gene, whose product is membrane bound, or of the cat gene, whose product is cytosolic. Putting a second copy of the Tac promoter downstream from lacZ, approximately 3,000 bp from leu-500 in the same orientation as the latter, resulted in 30-fold increase in lacZ expression upon isopropyl-beta-D-thiogalactopyranoside induction. Again, these effects were independent of the nature of the gene upstream from leu-500 (tet or cat). With both tet- and cat-harboring constructs, activation of the two Tac promoter copies caused plasmid DNA to become hypernegatively supercoiled in topA mutant cells. Thus, neither leu-500 activation nor hypernegative plasmid DNA supercoiling appears to require membrane anchoring of DNA in this system. Replacing the downstream copy of Tac with a constitutive promoter resulted in high-level lacZ expression even when the upstream copy was repressed. Under these conditions, no hypernegative DNA supercoiling was observed, indicating that the activity of plasmid-borne leu-500 in topA mutant cells does not necessarily correlate with the linking deficit of plasmid DNA. The response of the leu-500-lacZ fusion to downstream transcription provides a sensitive assay for transcriptional supercoiling in bacteria.

Molecular genetic analysis of the mldr mouse: a spontaneous revertant at the mld locus containing a recombinant myelin basic protein gene.

The mld mutation is a complex genetic lesion affecting the myelin basic protein (MBP) locus in the mouse. The mutation consists of a variety of DNA rearrangements including: tandem duplication of the MBP structural gene, partial inversion of the 3' end of the upstream gene copy, duplication of a region flanking the rearrangement junction in the upstream copy and insertion between the two gene copies of a segment of extraneous DNA not associated with the wild-type MBP locus. The net result of the mutation is a dysfunctional MBP locus. Homozygous mld/mld mice produce very little MBP and consequently very little myelin. They exhibit a clinical phenotype characteristic of hypomyelination (shaking, convulsions). We have discovered a revertant mld mouse which does not exhibit clinical symptoms of hypomyelination. Genetic analysis indicates that the reversion is allelic to mld. We have designated the revertant locus mldr. Restriction analysis of mldr genomic DNA indicates that there is a single intact MBP gene. Analysis of various junction regions using the polymerase chain reaction indicates that the single MBP gene in mldr is derived by recombination from the 5' end of the upstream gene and the 3' end of the downstream gene. Studies on MBP expression in mldr mice indicate that the developmental regulation, level of expression and pattern of post-transcriptional processing of MBP gene products in mldr are similar to wild type. These results indicate that the recombinant MBP gene in mldr is fully functional. From this we infer that the MBP-deficient phenotype of the original mld mutant is attributable to the complex rearrangements in the upstream gene copy which render the locus dysfunctional.(ABSTRACT TRUNCATED AT 250 WORDS)

Chimeric and Molecular Genetic Analysis of Myelin‐Deficient (Shiverer and Mld) Mutant Mice

The shiverer and myelin-deficient (mld) mutants are two allelic mutations. Both are characterized by hypomyelination in the CNS and deficient expression of the MBP gene. Chimeric analysis of the pathogenesis of shiverer showed that shiverer mutation acts intrinsic to the oligodendrocyte, which is the only cell type expressing the MBP gene in the CNS. Molecular genetic studies by several groups demonstrated that shiverer is a deletion mutation in the MBP gene. Consequently, no MBP is produced in the shiverer mutant. In mld, however, partial expression of the MBP gene was observed. Interestingly, MBP was expressed in a mosaic fashion in the CNS of mld mice. Molecular genetic studies revealed that the mld mutant has duplicated MBP genes in tandem on a distal part (E2-4) of chromosome 18q. The reduced expression was based on the level of mRNA. A large portion is inverted in the upstream copy, and an intact copy is located downstream. We showed in mld mutants that antisense RNA corresponding to the inverted segment is transcribed, and it forms RNA duplex, with the RNA transcribed from the normal gene located downstream.

Repetitive sequence involvement in the duplication and divergence of mouse lysozyme genes.

Mouse M and P lysozymes are the products of separate genes, are specifically expressed in separate tissues, and are adapted to different functions. The lysozyme genes have assumed these markedly different characteristics following their generation by gene duplication 30-50 million years ago. The discovery of the lysozyme P gene only 5 kb upstream from the M gene in tandem repeat has enabled an investigation of the molecular basis of their duplication and subsequent divergence. The duplication is shown to have involved recombination between two B2 repeat sequences flanking the original gene. The resulting downstream copy has retained the myeloid specificity of expression along with just 1.7 kb of upstream sequences, while the upstream copy is inactive in macrophages and has become expressed instead in the small intestine. Although multiple gene conversion events have served to maintain a generally high homology between the genes, certain regions have been found to be specific for either one of the gene pair: two repetitive sequences peculiar to the P region may serve to protect the coding regions from gene conversion, while sequences unique to the M gene may be more directly involved in differential regulation.

Detailed structural analysis of a genome rearrangement in bovine rotavirus

A genome rearrangement involving RNA segment 11 of a bovine rotavirus has been analysed by molecular cloning and sequencing. This revealed that the rearranged genome segment was generated by a head to tail concatemerisation of two almost full length copies of segment 11. The upstream copy of the gene has lost its 3' end and the downstream copy its 5' end. The truncation of the upstream copy of the gene occurs within the termination codon for VP11 converting it from a UAG to a UGA, the rearranged gene is therefore still able to encode a normal VP11. The possible mechanisms by which this rearrangement may have been generated are discussed.

Binding sites of HeLa cell nuclear proteins on the upstream region of adenovirus type 5 E1A gene

Twenty one binding sites of HeLa cell nuclear proteins were identified on the upstream region of adenovirus type 5 E1A gene using DNase I footprint assay. The proximal promoter region contained five binding sites that overlapped the cap site, TATA box, TATA-like sequence, CCAAT box, and -100 region relative to the E1A cap site(+1). The -190 region was a potential site for octamer-motif binding proteins, such as NFIII and OBP100. An upstream copy of the E1A enhancer element 1 was the site for a factor (E1A-F) with the binding specificity of XGGAYGT (X = A, C; Y = A, T). E1A-F factor also bound to three other sites, one of which coincided with the distal E1A enhancer element. The distal element also contained a potential site for ATF factor. The adenovirus minimal origin of DNA replication competed for DNA-protein complex formation on the CCAAT and TATA box region and the -190 region, suggesting that these regions interacted with a common or related factor.

Recombination within the upstream gene of duplicated myelin basic protein genes of myelin deficient shimld mouse results in the production of antisense RNA.

The myelin deficient shimld mouse is an autosomal recessive mutant, characterized by hypomyelination in the central nervous system. The expression of the myelin basic protein (MBP) gene is inhibited transcriptionally. The MBP gene is duplicated tandemly in mld, and exons 3 to 7 of the upstream copy is inverted. In the present studies, we determined the approximate position of the 5' boundary and the nucleotide sequence surrounding the 3' boundary of the inversion and found a number of sequences homologous to the switching regions of mouse immunoglobulin heavy chain gene and J regions of human T cell receptor genes. Antisense RNA complementary to exons 3 and 7, which correspond to the inverted segment, was detected by RNase protection studies. This abnormal transcript was also shown to elongate through the inverted segment to reach the transcription initiation site of the downstream gene.