Diverse Genetic Elements Research Articles

Detecting Class 1 Integrons and Their Variable Regions in Escherichia coli Whole-Genome Sequences Reported from Andean Community Countries.

Various genetic elements, including integrons, are known to contribute to the development of antimicrobial resistance. Class 1 integrons have been identified in E. coli isolates and are associated with multidrug resistance in countries of the Andean Community. However, detailed information on the gene cassettes located on the variable regions of integrons is lacking. Here, we investigated the presence and diversity of class 1 integrons, using an in silico approach, in 2533 whole-genome sequences obtained from EnteroBase. IntFinder v1.0 revealed that almost one-third of isolates contained these platforms. Integron-bearing isolates were associated with environmental, food, human, and animal origins reported from all countries under scrutiny. Moreover, they were identified in clones known for their pathogenicity or multidrug resistance. Integrons carried cassettes associated with aminoglycoside (aadA), trimethoprim (dfrA), cephalosporin (blaOXA; blaDHA), and fluoroquinolone (aac(6')-Ib-cr; qnrB) resistance. These platforms showed higher diversity and larger numbers than previously reported. Moreover, integrons carrying more than three cassettes in their variable regions were determined. Monitoring the prevalence and diversity of genetic elements is necessary for recognizing emergent patterns of resistance in pathogenic bacteria, especially in countries where various factors are recognized to favor the selection of resistant microorganisms.

Molecular Characterization of Class A-ESBLs, Class B-MBLs, Class C-AmpC, and Class D-OXA Carbapenemases in MDR Acinetobacter baumannii Clinical Isolates in a Tertiary Care Hospital, West Bengal, India.

Background Acinetobacter calcoaceticus baumannii (ACB) complex has become a major concern nowadays because of its increasing involvement in several severe infections associated with catheter-related bloodstream and urinary tract infections, ventilator-associated pneumonia, cerebrospinal shunt-related meningitis, and wound infections. Multiple drug-resistant (MDR) ACB cases have been described with an increasing trend where at least it is resistant to a minimum of three antimicrobial groups. The mortality rate associated with A. baumannii is significantly higher than all Acinetobacterspp. isolates with the most prevalence seen in India and Thailand. The rapid spread of high resistance to most potent antimicrobial drugs is due to its ability to incorporate resistance determinants despite multifactorial reasons such as alteration in permeability of cell membrane by either losing expression of outer membrane porins or excess production of efflux pumps. This study aims to characterize resistance determinants responsible for MDR at the genetic level and emphasizes the use of genotyping in routine diagnosis as genotype analysis is reliable and valid. Methodology A total of 289 ACB complex clinical isolates were included in this study. The study for species-level identification of A. baumannii was conducted at the Department of Microbiology, IQ City Medical College Hospital, Durgapur, West Bengal. In addition, the detection of encoded genes associated with class A-extended spectrum beta-lactamases (i.e., CTX-M, KPC, SHV, and TEM genes), class B-metallo-β-lactamases (i.e., IMP, NDM, and VIM genes), Class C-AmpC cephalosporinase, and classD-OXA carbapenemases (i.e., blaOXA-10/11, blaOXA-24, blaOXA-48, blaOXA-58, blaOXA-143, and blaOXA-235was done using real-time polymerase chain reaction. Results All 289 non-repetitive ACB complex clinical isolates were confirmed as A. baumannii, of which 277 (96%) isolates were MDR. There were no findings of blaCTX-M, blaKPC, blaSHV, blaTEM, blaIMP, blaVIM, blaAmpC, blaOXA-10/11, blaOXA-24, blaOXA-48, blaOXA-58, blaOXA-143, and blaOXA-235 genes in our study. However, there were four (1.44%) positive findings of the blaNDM gene. All MDR isolates (n = 277) were positive for the blaOXA-51 gene. In addition, blaOXA-23 was positive in 269 (97.12%) isolates. Conclusions The oxacillinase production corresponding to blaOXA-23 and blaOXA-51 were the most prevalent antibiotic resistance determinants among MDR A. baumanniiin our study. Four (1.44%) isolates had the multiple genes blaOXA-51, blaOXA-23, and blaNDM that shows the coexistence of diverse genetic elements involved in MDR A. baumannii, resulting in total resistance except for a few potent drugs such as colistin and tigecycline. Genotyping is helpful in determining the contribution of the isolates in understanding their association with encoded genes, which, in turn, helps in designing effective surveillance and control strategies in the management of such MDR isolates.

Genomic Island-Encoded Histidine Kinase and Response Regulator Coordinate Mannose Utilization with Virulence in Enterohemorrhagic Escherichia coli.

Enterohemorrhagic Escherichia coli (EHEC) is a highly adaptive pathogen and has acquired diverse genetic elements, such as genomic islands and prophages, via horizontal gene transfer to promote fitness in vivo. Two-component signaling systems (TCSs) allow bacteria to sense, respond to, and adapt to various environments. This study identified a putative two-component signaling system composed of the histidine kinase EDL5436 (renamed LmvK) and the response regulator EDL5428 (renamed LmvR) in EHEC. lmvK and lmvR along with EDL5429 to EDL5434 (EDL5429-5434) between them constitute the OI167 genomic island and are highly associated with the EHEC pathotype. EDL5429-5434 encode transporters and metabolic enzymes that contribute to growth on mannose and are directly upregulated by LmvK/LmvR in the presence of mannose, as revealed by quantitative PCR (qPCR) and DNase I footprint assays. Moreover, LmvR directly activates the expression of the type III secretion system in response to mannose and promotes the formation of attaching and effacing lesions on HeLa cells. Using human colonoid and mouse infection models, we show that lmvK and lmvR contributed greatly to adherence and microcolony (MC) formation ex vivo and colonization in vivo. Finally, RNA sequencing and chromatin immunoprecipitation coupled with sequencing analyses identified additional direct targets of LmvR, most of which are involved in metabolism. Given that mannose is a mucus-derived sugar that induces virulence and is preferentially used by EHEC during infection, our data revealed a previously unknown mechanism by which EHEC recognizes the host metabolic landscape and regulates virulence expression accordingly. Our findings provide insights into how pathogenic bacteria evolve by acquiring genetic elements horizontally to adapt to host environments. IMPORTANCE The gastrointestinal tract represents a complex and challenging environment for enterohemorrhagic Escherichia coli (EHEC). However, EHEC is a highly adaptable pathogen, requiring only 10 to 100 CFUs to cause infection. This ability was achieved partially by acquiring mobile genetic elements, such as genomic islands, that promote overall fitness. Mannose is an intestinal mucus-derived sugar that stimulates virulence and is preferentially used by EHEC during infection. Here, we characterize the OI167 genomic island of EHEC, which encodes a novel two-component signaling system (TCS) and transporters and metabolic enzymes (EDL5429-5434) involved in mannose utilization. The TCS directly upregulates EDL5429-5434 and genes encoding the type III secretion system in the presence of mannose. Moreover, the TCS contributes greatly to EHEC virulence ex vivo and in vivo. Our data demonstrate an elegant example in which EHEC strains evolve by acquiring genetic elements horizontally to recognize the host metabolic landscape and regulate virulence expression accordingly, leading to successful infections.

Contamination of Proteus mirabilis harbouring various clinically important antimicrobial resistance genes in retail meat and aquatic products from food markets in China.

Proteus mirabilis is an opportunistic pathogen frequently associated with nosocomial infection and food poisoning cases. Contamination of P. mirabilis in retail meat products may be important transmission routes for human infection with P. mirabilis. In this study a total of 89 P. mirabilis strains were isolated from 347 samples in 14 food markets in China and subjected to whole-genome sequencing. Phylogenetic analysis showed that all 89 strains were divided into 81 different clones (SNPs >5), indicating high genetic diversity of P. mirabilis in food markets. Antimicrobial susceptibility testing showed that 81 (91.01%) strains displayed multidrug resistance profiles. Seventy-three different resistance genes (or variants) were found, including various clinically important antimicrobial resistance genes aac(6')-Ib-cr (77.53%), bla CTX-M (39.33%), fosA3 (30.34%), as well as multiresistance gene cfr (4.50%), tigecycline resistance gene cluster tmexCD3-toprJ1 (4.50%) and carbapenemase gene bla NDM-1 (1.12%). Diverse genetic elements including Tn7 transposon, plasmid, SXT/R391 integrative conjugative element were associated with the horizontal transfer of cfr. tmexCD3-toprJ1 and bla NDM-1 were located on ICEPmiChnJZ26 and Salmonella genomic island 1, respectively. Our study emphasized high contamination of P. mirabilis harbouring various clinically important antimicrobial resistance genes in retail meat and aquatic products, which might be an important issue in terms of food safety and human health.

Various mobile genetic elements carrying optrA in Enterococcus faecium and Enterococcus faecalis isolates from swine within the same farm.

In this study, the distribution of the oxazolidinone/phenicol resistance gene optrA and the mobile genetic elements involved in its dissemination were analysed among enterococcal isolates from a farrow-to-finish swine farm. Enterococcus faecium and Enterococcus faecalis isolates were obtained from all pig production stages in the farm. The optrA-carrying E. faecium and E. faecalis isolates were subjected to PFGE and antimicrobial susceptibility testing. Complete sequences of the genetically unrelated optrA-carrying E. faecium and E. faecalis isolates were determined using Illumina HiSeq and MinION platforms. The optrA gene was present in 12.2% (23/188) of the E. faecium and E. faecalis isolates, most of which originated from nursery and finishing stages. The 23 optrA-positive Enterococcus isolates represented 15 PFGE types. WGS of representative isolates of the 15 PFGE types showed that optrA was carried by diverse genetic elements either located in the chromosomal DNA or on plasmids. A novel optrA-bearing genetic element was identified on two distinct multi-resistance plasmids from E. faecium. Two new hybrid plasmids carrying several resistance genes were found in two E. faecalis isolates. pC25-1-like plasmids and chromosomally integrated Tn6674 and Tn6823-like transposons were prevalent in the remaining Enterococcus isolates. The gene optrA was found in genetically unrelated E. faecium and E. faecalis isolates from the same farm. Analysis of the genetic contexts of optrA suggested that horizontal transfer including different plasmids and transposons played a key role in the dissemination of optrA in this farm.

Proteomics technique opens new frontiers in mobilome research.

ABSTRACTA large proportion of the genome of most eukaryotic organisms consists of highly repetitive mobile genetic elements. The sum of these elements is called the “mobilome,” which in eukaryotes is made up mostly of transposons. Transposable elements contribute to disease, evolution, and normal physiology by mediating genetic rearrangement, and through the “domestication” of transposon proteins for cellular functions. Although ‘omics studies of mobilome genomes and transcriptomes are common, technical challenges have hampered high-throughput global proteomics analyses of transposons. In a recent paper, we overcame these technical hurdles using a technique called “proteomics informed by transcriptomics” (PIT), and thus published the first unbiased global mobilome-derived proteome for any organism (using cell lines derived from the mosquito Aedes aegypti). In this commentary, we describe our methods in more detail, and summarise our major findings. We also use new genome sequencing data to show that, in many cases, the specific genomic element expressing a given protein can be identified using PIT. This proteomic technique therefore represents an important technological advance that will open new avenues of research into the role that proteins derived from transposons and other repetitive and sequence diverse genetic elements, such as endogenous retroviruses, play in health and disease.

Biochemical, Virulence and Resistance Features in Bacterial Strains Recovered from Hospital Surfaces after Decontamination with Quaternary Ammonium Compounds, Triclosan and Iodine Desinfectatnts

The purpose of this study was to investigate the resistance and virulence markers in microbial strains isolated from the hospital environment, recovered after surfaces decontamination with quaternary ammonium compounds, triclosan, iodine disinfectants, in order to predict their role in healthcare-associated infections. The resistance phenotypes were established using disk diffusion and double-disk diffusion test. Simplex and multiplex PCR assays were used to identify the genetic support of cell-associated and soluble virulence factors The Gram-negative bacterial strains isolated from hospital surfaces after decontamination were resistant in high proportions to beta-lactam antibiotics, including penicillins and associations with beta-lactamase inhibitors, third and fourth generation cephalosporins and carbapenems (encoded by blaTEM, blaNDMlike blaCTX-Mlike, blaOXA-48like genes), quinolones (QnrA, gyrB, parE genes), aminoglycosides (aac3Ia gene), and tetracyclines. Regarding the virulence profiles, P. aeruginosa strains revealed the plcH gene (86.6%) followed by plcH, ExoS, algD and ExoU, while the Enterobacteriaceae strains, fimH (24%), followed by papC, sfaDE, hlyA, cnf1, eaea and VT2 genes. S. aureus isolates revealed the SCCmec cassette of type II (40%), followed by type V, IIIA and type IV (2B) and the clfA, clfB, and can adhesion genes. These findings demonstrate that incorrect practices in biocides use, regarding contact time and concentrations or instruments and surfaces insufficient cleaning before disinfection can decrease their effect and favor the persistence of resistant and virulent microbial strains in the hospital environment. The multiple drug resistance and virulence determinants, encoded by diverse genetic elements suggest the potential of these strains to persist and initiate hospital-associated infections.

Genome sequences of copper resistant and sensitive Enterococcus faecalis strains isolated from copper-fed pigs in Denmark.

Six strains of Enterococcus faecalis (S1, S12, S17, S18, S19 and S32) were isolated from copper fed pigs in Denmark. These Gram-positive bacteria within the genus Enterococcus are able to survive a variety of physical and chemical challenges by the acquisition of diverse genetic elements. The genome of strains S1, S12, S17, S18, S19 and S32 contained 2,615, 2,769, 2,625, 2,804, 2,853 and 2,935 protein-coding genes, with 41, 42, 27, 42, 32 and 44 genes encoding antibiotic and metal resistance, respectively. Differences between Cu resistant and sensitive E. faecalis strains, and possible co-transfer of Cu and antibiotic resistance determinants were detected through comparative genome analysis.

Comparative genomics of IncP-1ε plasmids from water environments reveals diverse and unique accessory genetic elements

The goal of this study was to determine and compare the complete genome sequences of three new broad-host-range conjugative plasmids. Plasmids pMLUA1, pMLUA3 and pMLUA4 were previously recovered from estuarine water by exogenous plasmid isolation and ranged in size from ∼55 to 59kb. Comparative genomics showed that their backbone region was identical to the prototype pKJK5 and other IncP1-ε plasmids captured from soils. The accessory region was inserted between the tra region and parA, and presented the typical IncP-1ε ISPa17 and Tn402-like transposon modules. Nevertheless, new class 1 integrons were identified (In794, carrying aadA5 and In795, carrying qacF5-aadA5), as well as a composite transposon IS26-msr(E)-mph(E)-IS26 carrying genes that confer resistance to macrolides. A new insertion sequence, termed ISUnCu17, was also identified on pMLUA3. The architecture of the accessory regions implies the occurrence of multiple insertions and deletions. These data support the notion that IncP-1 plasmids from the ε subgroup are proficient in the capture of diverse genetic elements, including antibiotic resistance genes, and thus may contribute to the co-selection of several resistance determinants. This study constitutes the first report of completely sequenced IncP-1ε plasmids from water environments, and enhances our understanding of the geographic distribution and genetic diversity of these replicons.

Neutral Theory Predicts the Relative Abundance and Diversity of Genetic Elements in a Broad Array of Eukaryotic Genomes

It is universally true in ecological communities, terrestrial or aquatic, temperate or tropical, that some species are very abundant, others are moderately common, and the majority are rare. Likewise, eukaryotic genomes also contain classes or “species” of genetic elements that vary greatly in abundance: DNA transposons, retrotransposons, satellite sequences, simple repeats and their less abundant functional sequences such as RNA or genes. Are the patterns of relative species abundance and diversity similar among ecological communities and genomes? Previous dynamical models of genomic diversity have focused on the selective forces shaping the abundance and diversity of transposable elements (TEs). However, ideally, models of genome dynamics should consider not only TEs, but also the diversity of all genetic classes or “species” populating eukaryotic genomes. Here, in an analysis of the diversity and abundance of genetic elements in >500 eukaryotic chromosomes, we show that the patterns are consistent with a neutral hypothesis of genome assembly in virtually all chromosomes tested. The distributions of relative abundance of genetic elements are quite precisely predicted by the dynamics of an ecological model for which the principle of functional equivalence is the main assumption. We hypothesize that at large temporal scales an overarching neutral or nearly neutral process governs the evolution of abundance and diversity of genetic elements in eukaryotic genomes.

Large-Scale Analysis of Plasmid Relationships through Gene-Sharing Networks

Plasmids are vessels of genetic exchange in microbial communities. They are known to transfer between different host organisms and acquire diverse genetic elements from chromosomes and/or other plasmids. Therefore, they constitute an important element in microbial evolution by rapidly disseminating various genetic properties among different communities. A paradigmatic example of this is the dissemination of antibiotic resistance (AR) genes that has resulted in the emergence of multiresistant pathogenic bacterial strains. To globally analyze the evolutionary dynamics of plasmids, we built a large graph in which 2,343 plasmids (nodes) are connected according to the proteins shared by each other. The analysis of this gene-sharing network revealed an overall coherence between network clustering and the phylogenetic classes of the corresponding microorganisms, likely resulting from genetic barriers to horizontal gene transfer between distant phylogenetic groups. Habitat was not a crucial factor in clustering as plasmids from organisms inhabiting different environments were often found embedded in the same cluster. Analyses of network metrics revealed a statistically significant correlation between plasmid mobility and their centrality within the network, providing support to the observation that mobile plasmids are particularly important in spreading genes in microbial communities. Finally, our study reveals an extensive (and previously undescribed) sharing of AR genes between Actinobacteria and Gammaproteobacteria, suggesting that the former might represent an important reservoir of AR genes for the latter.

Characterisation of the CTX-M-15-encoding gene in Klebsiella pneumoniae strains from the Barcelona metropolitan area: plasmid diversity and chromosomal integration

The localisation and genetic organisation of bla CTX-M-15 were studied in 37 CTX-M-15-producing Klebsiella pneumoniae isolates collected from 2005 to 2008 within the Barcelona metropolitan area. Polymerase chain reaction (PCR)-based replicon typing and Southern hybridisations were used to identify the bla CTX-M-15 location. The genetic environment was analysed by PCR mapping and sequencing, and transferability of bla CTX-M-15 was evaluated by conjugation and transformation assays. The majority of the 37 isolates carried bla CTX-M-15 in a plasmid location, frequently associated with the aac(6′)-Ib-cr gene. Plasmids encoding bla CTX-M-15 carried three distinct replicons, i.e. IncFII, IncR and IncFIIk, the latter two not having been described previously in association with bla CTX-M-15. Several of these plasmids were not self-transferable. Furthermore, in all isolates belonging to sequence type ST-1, bla CTX-M-15 was found integrated into the K. pneumoniae chromosome. In all the studied isolates, the mobile element IS Ecp1 was found upstream of bla CTX-M-15, whereas IS 26 was found inserted within IS Ecp1 in several isolates, in previously unreported positions. In conclusion, these findings indicate that among K. pneumoniae strains isolated in the Barcelona metropolitan area, bla CTX-M-15 is associated with diverse genetic elements, including the IncR and IncFIIk replicons, as reported for the first time here, and the chromosome.

International Spread and Persistence of TEM-24 Is Caused by the Confluence of Highly Penetrating Enterobacteriaceae Clones and an IncA/C 2 Plasmid Containing Tn 1696 ::Tn 1 and IS 5075 -Tn 21

TEM-24 remains one of the most widespread TEM-type extended-spectrum beta-lactamases (ESBLs) among Enterobacteriaceae. To analyze the reasons influencing its spread and persistence, a multilevel population genetics study was carried out on 28 representative TEM-24 producers from Belgium, France, Portugal, and Spain (13 Enterobacter aerogenes isolates, 6 Escherichia coli isolates, 6 Klebsiella pneumoniae isolates, 2 Proteus mirabilis isolates, and 1 Klebsiella oxytoca isolate, from 1998 to 2004). Clonal relatedness (XbaI pulsed-field gel electrophoresis [PFGE] and E. coli phylogroups) and antibiotic susceptibility were determined by standard procedures. Plasmid analysis included determination of the incompatibility group (by PCR, hybridization, and/or sequencing) and comparison of restriction fragment length polymorphism (RFLP) patterns. Characterization of genetic elements conferring antibiotic resistance included integrons (classes 1, 2, and 3) and transposons (Tn3, Tn21, and Tn402). Similar PFGE patterns were identified among E. aerogenes, K. pneumoniae, and P. mirabilis isolates, while E. coli strains were diverse (phylogenetic groups A, B2, and D). Highly related 180-kb IncA/C2 plasmids conferring resistance to kanamycin, tobramycin, chloramphenicol, trimethoprim, and sulfonamides were identified. Each plasmid contained defective In0-Tn402 (dfrA1-aadA1, aacA4, or aacA4-aacC1-orfE-aadA2-cmlA1) and In4-Tn402 (aacA4 or dfrA1-aadA1) variants. These integrons were located within Tn21, Tn1696, or hybrids of these transposons, with IS5075 interrupting their IRtnp and IRmer. In all cases, blaTEM-24 was part of an IS5075-DeltaTn1 transposon within tnp1696, mimicking other genetic elements containing blaTEM-2 and blaTEM-3 variants. The international dissemination of TEM-24 is fuelled by an IncA/C2 plasmid acquired by different enterobacterial clones which seem to evolve by gaining diverse genetic elements. This work highlights the risks of a confluence between highly penetrating clones and highly promiscuous plasmids in the spread of antibiotic resistance, and it contributes to the elucidation of the origin and evolution of TEM-2 ESBL derivatives.

Analysis of CRE‐mediated recombination driven by myosin light chain 1/3 regulatory elements in embryonic and adult skeletal muscle: A tool to study fiber specification

An increasing number of genes have been implicated in skeletal muscle fiber diversity. To study the contribution of diverse genetic elements to the regulation of fiber-type composition, we generated a transgenic mouse in which CRE recombinase expression is driven by muscle-specific regulatory sequences of the myosin light chain 1/3 locus (MLC). Using ROSA26 conditional reporter mice, we detected expression of the MLC-Cre transgene starting from embryonic day 12.5 (E12.5). By E15, recombination was detected in all muscle-derived structures. Immunohistochemical analysis revealed CRE activity was restricted to fast-twitch (type II) and excluded from slow-twitch (type I) fibers of skeletal muscle. The MLC-Cre transgenic mouse can be used in conjunction with conditional alleles to study both developmental patterning and maintenance of fast fiber-type phenotypes.

Immunoglobulin gene sequence analysis of anti-cardiolipin and anti-cardiolipin idiotype (H3) human monoclonal antibodies.

Heavy and light chain variable region nucleotide sequences were derived from 6 human hybridoma antibodies which bear characteristics of antibodies associated with the phospholipid antibody syndrome. All antibodies originated from non-autoimmune individuals and were polyspecific. Four of these reacted with cardiolipin (and other antigens) and three carried the H3 idiotype which is expressed on a high percentage of disease-associated anti-cardiolipin antibodies. This idiotype was localized to the lambda light chain of the H3 monoclonal antibody and found on two other antibodies which like H3 expressed V lambda 4 or the related V lambda 3 subgroup light chains. The H3 idiotype however did not define these subgroups nor was it required or sufficient for anti-cardiolipin activity. Anti-cardiolipin binding was found in VH1, VH3 and VH4 heavy chain families and in a V kappa 1 light chain. The D region was diverse in both length and gene usage. Although all cardiolipin binding antibodies showed little deviation from germline variable (V) gene sequences, where mutations occurred they tended to be replacement mutations and clustered in complementarity determining regions (CDR) suggesting these B cells were derived from antigen-driven responses. These results from our panel of hybridomas and their comparison to other human antibodies provide extensive information on the diversity of genetic elements which can be used by cardiolipin-binding antibodies. We also show gene sequences which encode the disease-associated H3 idiotype and its location on lambda light chains, which imply that some labda light chains may be preferentially utilized in auto-reactive hybridomas.

Phosphorylation influences the binding of the yeast RAP1 protein to the upstream activating sequence of thePGKgene

Yeast repressor activator protein 1 (RAP1) binds in vitro to specific DNA sequences that are found in diverse genetic elements. Expression of the yeast phosphoglycerate kinase gene (PGK) requires the binding of RAP1 to the activator core sequence within the upstream activating sequence (UAS) of PGK. A DNA fragment Z+ which contains the activator core sequence of the PGK(UAS) has been shown to bind RAP1. Here we report that phosphatase treatment of RAP1 affected its binding to the PGK(UAS) but that this depended on the nature of the sequence flanking the 5' end of the activator core sequence. When the sequence flanking the 5' end of the activator core sequence was different from the PGK RAP1-binding site, phosphatase treatment of RAP1 decreased its binding to the DNA. When the 5' end of the binding site was a match to the PGK RAP1-binding site dephosphorylation of RAP1 increased RAP1 binding to the DNA. These observations were reproduced when the minimal functional DNA-binding domain of the RAP1 protein was used, implicating a phosphorylation-dependent binding of RAP1. This is the first evidence for phosphorylation-dependent binding of RAP1.

Connections between transcriptional activators, silencers, and telomeres as revealed by functional analysis of a yeast DNA-binding protein.

General regulatory factor I (GRFI) is a yeast protein that binds in vitro to specific DNA sequences at diverse genetic elements. A strategy was pursued to test whether GRFI functions in vivo at the sequences bound by the factor in vitro. Matches to a consensus sequence for GRFI binding were found in a variety of locations: upstream activating sequences (UASs), silencers, telomeres, and transcribed regions. All occurrences of the consensus sequence bound both crude and purified GRFI in vitro. All binding sites for GRFI, regardless of origin, provided UAS function in test plasmids. Also, GRFI binding sites specifically stimulated transcription in a yeast in vitro system, indicating that GRFI can function as a positive transcription factor. The stimulatory effect of GRFI binding sites at UASs for the PYK1 and ENO1 genes is significantly enhanced by flanking DNA elements. By contrast, regulatory sequences that flank the GRFI binding site at HMR E convert this region to a transcriptional silencer.

Connections between transcriptional activators, silencers, and telomeres as revealed by functional analysis of a yeast DNA-binding protein.

General regulatory factor I (GRFI) is a yeast protein that binds in vitro to specific DNA sequences at diverse genetic elements. A strategy was pursued to test whether GRFI functions in vivo at the sequences bound by the factor in vitro. Matches to a consensus sequence for GRFI binding were found in a variety of locations: upstream activating sequences (UASs), silencers, telomeres, and transcribed regions. All occurrences of the consensus sequence bound both crude and purified GRFI in vitro. All binding sites for GRFI, regardless of origin, provided UAS function in test plasmids. Also, GRFI binding sites specifically stimulated transcription in a yeast in vitro system, indicating that GRFI can function as a positive transcription factor. The stimulatory effect of GRFI binding sites at UASs for the PYK1 and ENO1 genes is significantly enhanced by flanking DNA elements. By contrast, regulatory sequences that flank the GRFI binding site at HMR E convert this region to a transcriptional silencer.

Neuronal Expression of chimeric genes in transgenic mice

Gene expression may occur in unexpected ectopic sites when diverse genetic elements are juxtaposed as chimeric genes in transgenic mice. To determine the specific contribution of the promoter and reporter gene in ectopic expression, we have analyzed the expression of 14 different fusion genes in transgenic mice. Chimeric genes containing the mouse metallothionein-I promoter linked to either the rat or human growth hormone gene or the calcitonin/CGRP gene are expressed in a very similar pattern of neuronal regions. This ectopic expression is not a unique feature of the metallothionein promoter, since transferring the human growth hormone gene to four other heterologous promoters resulted in varying degrees of ectopic expression in overlapping subsets of cortical and hypothalamic neurons. The novel pattern of ectopic expression suggests that these otherwise unrelated neurons share a common developmental regulatory machinery for activation of gene transcription.

Human terminal deoxyribonucleotidyltransferase: molecular cloning and structural analysis of the gene and 5' flanking region.

Human terminal deoxyribonucleotidyltransferase (nucleoside-triphosphate:DNA deoxynucleotidylexotransferase, EC 2.7.7.31) cDNA contains an open reading frame of 1530 base pairs (bp) corresponding to a protein containing 510 amino acids. The encoded protein is a template-independent DNA polymerase found only in a restricted population of normal and malignant prelymphocytes. To begin to investigate the genetic elements responsible for the tissue-specific expression of terminal deoxyribonucleotidyltransferase, genomic clones containing the entire human gene were isolated and characterized. Initially, cDNA clones were isolated from a library generated from the human lymphoblastoid cell line, MOLT-4R. A cDNA clone containing the entire coding region of the protein was used to isolate a series of overlapping clones from two human genomic libraries. The gene comprises 11 exons and 10 introns and spans 49.4 kilobases. The 5' flanking region (709 bp) including exon 1 was sequenced. Several putative transcription initiation sites were mapped. Within 500 nucleotides of the translation start site, a series of promoter elements was detected. "TATA" and "CAAT" sequences, respectively, were found to start at nucleotides -185 and -204, -328, and 465 and -505. Start sites were found for a cyclic AMP-dependent promoter analog at nucleotide -121, an eight-base sequence corresponding to the IgG promoter enhancer (cd) at nucleotide -455, and an analog of the IgG promoter (pd) at nucleotide -159. These findings suggest that transcripts coding for terminal deoxyribonucleotidyltransferase may be variable in length and that transcription may be influenced by a variety of genetic elements.