Combination Of Multiple Mutations Research Articles

Resistance to 16-Membered Macrolides, Tiamulin and Lincomycin in a Swine Isolate of Acholeplasma laidlawii.

Acholeplasma (A.) laidlawii is an opportunistic pathogen with the ability to disseminate resistance determinants to antibiotics; however, its resistance to macrolides has been less studied. The aim of the present study was to characterize the mechanisms responsible for the resistance to macrolides, tiamulin and lincomycin found in a strain of A. laidlawii isolated from a pig with pneumonia. MICs of erythromycin, 15- and 16-membered macrolides, tiamulin and lincomycin were determined by microdilution method with and without reserpine, an inhibitor of ABC efflux pumps and regions of the genome were sequenced. Reserpine only decreased lincomycin MIC but it did not change the MICs of macrolides and tiamulin. The analysis of the DNA sequence of 23S rRNA showed nucleotide substitutions at eight different positions, although none of them were at positions previously related to macrolide resistance. Five mutations were found in the L22 protein, one of them at the stop codon. In addition, two mutations were found in the amino acid sequence of L4. The combination of multiple mutations in the ribosomal proteins L22 and L4 together with substitutions in 23S rRNA DNA sequence was associated with the resistance to macrolides, the pleuromutilin and lincomycin in the studied A. laidlawii strain.

Selection for novel metabolic capabilities in Salmonella enterica.

Bacteria are known to display extensive metabolic diversity and many studies have shown that they can use an extensive repertoire of small molecules as carbon‐ and energy sources. However, it is less clear to what extent a bacterium can expand its existing metabolic capabilities by acquiring mutations that, for example, rewire its metabolic pathways. To investigate this capability and potential for evolution of novel phenotypes, we sampled large populations of mutagenized Salmonella enterica to select very rare mutants that can grow on minimal media containing 124 low molecular weight compounds as sole carbon sources. We found mutants growing on 18 of these novel carbon sources, and identified the causal mutations that allowed growth for four of them. Mutations that relieve physiological constraints or increase expression of existing pathways were found to be important contributors to the novel phenotypes. For the remaining 14 novel phenotypes, whole genome sequencing of independent mutants and genetic analysis suggested that these novel metabolic phenotypes result from a combination of multiple mutations. This work, by virtue of identifying the genetic and mechanistic basis for new metabolic capabilities, sheds light on the properties of adaptive landscapes underlying the evolution of novel phenotypes.

Use of whole-genome sequencing to predict Mycobacterium tuberculosis drug resistance in Indonesia.

Whole-genome sequencing (WGS) is rarely used for drug resistance testing of Mycobacterium tuberculosis in high-endemic settings. Here we present the first study from Indonesia, which has the third highest tuberculosis (TB) burden worldwide, with <50% of drug-resistant cases currently detected. WGS was applied for strains from 322 human immunodeficiency virus (HIV)-negative adult TB patients. Phenotypic drug susceptibility testing (DST) was performed for a proportion of the patients. Using WGS, mutations associated with drug resistance to any TB drug were identified in 51 (15.8%) of the 322 patients, including 42 patients (13.0%) with no prior TB treatment (primary resistance). Eight isolates (2.5%) were multidrug-resistant (MDR) and one was extensively drug-resistant (XDR). Most mutations were found in katG (n=18), pncA (n=18), rpoB (n=10), fabG1 (n=9) and embB (n=9). Agreement of WGS-based resistance and phenotypic DST to first-line drugs was high for isoniazid and rifampicin but was lower for ethambutol and streptomycin. Drug resistance was more common in Indo-Oceanic lineage strains (37.5%) compared with Euro-American (18.2%) and East-Asian lineage strains (10.3%) (P=0.044), but combinations of multiple mutations were most common among East-Asian lineage strains (P=0.054). These data support the potential use of WGS for more rapid and comprehensive prediction of drug-resistant TB in Indonesia. Future studies should address potential barriers to implementing WGS, the distribution of specific resistance mutations, and the association of particular mutations with endemic M. tuberculosis lineages in Indonesia.

Reverse Engineering Field Isolates of Myxoma Virus Demonstrates that Some Gene Disruptions or Losses of Function Do Not Explain Virulence Changes Observed in the Field.

The coevolution of myxoma virus (MYXV) and wild European rabbits in Australia and Europe is a paradigm for the evolution of a pathogen in a new host species. Genomic analyses have identified the mutations that have characterized this evolutionary process, but defining causal mutations in the pathways from virulence to attenuation and back to virulence has not been possible. Using reverse genetics, we examined the roles of six selected mutations found in Australian field isolates of MYXV that fall in known or potential virulence genes. Several of these mutations occurred in genes previously identified as virulence genes in whole-gene knockout studies. Strikingly, no single or double mutation among the mutations tested had an appreciable impact on virulence. This suggests either that virulence evolution was defined by amino acid changes other than those analyzed here or that combinations of multiple mutations, possibly involving epistatic interactions or noncoding sequences, have been critical in the ongoing evolution of MYXV virulence. In sum, our results show that single-gene knockout studies of a progenitor virus can have little power to predict the impact of individual mutations seen in the field. The genetic determinants responsible for this canonical case of virulence evolution remain to be determined.IMPORTANCE The species jump of myxoma virus (MYXV) from the South American tapeti to the European rabbit populations of Australia and Europe is a canonical example of host-pathogen coevolution. Detailed molecular studies have identified multiple genes in MYXV that are critical for virulence, and genome sequencing has revealed the evolutionary history of MYXV in Australia and Europe. However, it has not been possible to categorically identify the key mutations responsible for the attenuation of or reversion to virulence during this evolutionary process. Here we use reverse genetics to examine the role of mutations in viruses isolated early and late in the Australian radiation of MYXV. Surprisingly, none of the candidate mutations that we identified as likely having roles in attenuation proved to be important for virulence. This indicates that considerable caution is warranted when interpreting the possible role of individual mutations during virulence evolution.

Trombozla başvuran pediatrik hastaların risk faktörleri açısından retrospektif olarak değerlendirilmesi: tek merkez deneyimi

Purpose: Thrombosis in children is a multifactorial disorder associated with both genetic and acquired factors. Idiopathic thrombosis is a rare condition. This study aimed to examine the etiology and risk factors of thrombosis in pediatric patients followed at Baskent University Adana Teaching and Research Center, a reference hospital in the southern part of Turkey. Material and Methods: This study included a sample of 49 pediatric patients who were retrospectively evaluated. The patient records were examined retrospectively in terms of age at diagnosis with thrombosis, gender, family history of thrombosis, localization of thrombosis, underlying primary disease and presence of catheter, as well as indicators of genetic risk factors. Results: The number of children presenting to our clinic during the study period was 10358. The incidence of thrombosis was calculated as 49 cases in 10358 children. The median age of the patients was 5.1 years (ranging from 0 to 17.2 years), and there were 32 (65.3%) males. In the analysis of the genetic factors causing susceptibility to primary thrombosis, only a portion of the patients were studied for genetic mutations. Among these, 5 patients showed factor V Leiden mutation, 13 patients MTHFR 1298C mutation, and 18 patients MTHFR 677CT mutation. Seven patients were found have a combination of multiple mutations. When we investigated any possible correlation between high lipoprotein (a) levels and the distribution of mutations, we found that 7 patients with mutations had normal levels of high lipoprotein (a), while other 10 patients with mutations were found to have elevated levels of lipoprotein (a). Conclusion: We conclude and strongly emphasize that lipoprotein (a) elevation is one of the important genetic risk factors, which might prove a major risk indicator, as valuable as other mutation screening tests performed by molecular genetics

Rift Valley Fever Virus MP-12 Vaccine Is Fully Attenuated by a Combination of Partial Attenuations in the S, M, and L Segments.

Rift Valley fever (RVF) is a mosquito-borne zoonotic disease endemic to Africa and characterized by a high rate of abortion in ruminants and hemorrhagic fever, encephalitis, or blindness in humans. RVF is caused by Rift Valley fever virus (RVFV; family Bunyaviridae, genus Phlebovirus), which has a tripartite negative-stranded RNA genome (consisting of the S, M, and L segments). Further spread of RVF into countries where the disease is not endemic may affect the economy and public health, and vaccination is an effective approach to prevent the spread of RVFV. A live-attenuated MP-12 vaccine is one of the best-characterized RVF vaccines for safety and efficacy and is currently conditionally licensed for use for veterinary purposes in the United States. Meanwhile, as of 2015, no other RVF vaccine has been conditionally or fully licensed for use in the United States. The MP-12 strain is derived from wild-type pathogenic strain ZH548, and its genome encodes 23 mutations in the three genome segments. However, the mechanism of MP-12 attenuation remains unknown. We characterized the attenuation of wild-type pathogenic strain ZH501 carrying a mutation(s) of the MP-12 S, M, or L segment in a mouse model. Our results indicated that MP-12 is attenuated by the mutations in the S, M, and L segments, while the mutations in the M and L segments confer stronger attenuation than those in the S segment. We identified a combination of 3 amino acid changes, Y259H (Gn), R1182G (Gc), and R1029K (L), that was sufficient to attenuate ZH501. However, strain MP-12 with reversion mutations at those 3 sites was still highly attenuated. Our results indicate that MP-12 attenuation is supported by a combination of multiple partial attenuation mutations and a single reversion mutation is less likely to cause a reversion to virulence of the MP-12 vaccine. Rift Valley fever (RVF) is a mosquito-transmitted viral disease that is endemic to Africa and that has the potential to spread into other countries. Vaccination is considered an effective way to prevent the disease, and the only available veterinary RVF vaccine in the United States is a live-attenuated MP-12 vaccine, which is conditionally licensed. Strain MP-12 is different from its parental pathogenic RVFV strain, strain ZH548, because of the presence of 23 mutations. This study determined the role of individual mutations in the attenuation of the MP-12 strain. We found that full attenuation of MP-12 occurs by a combination of multiple mutations. Our findings indicate that a single reversion mutation will less likely cause a major reversion to virulence of the MP-12 vaccine.

Sa1033 Interferon-Free Alisporivir Treatment Down-Regulates Interferon-Stimulated Genes Suggesting a Unique Antiviral Mechanism of Action for the Cyclophilin Inhibitor Alisporivir

BACKGROUND: VITAL-1 study assessed safety, tolerability and antiviral activity of the cyclophilin inhibitor alisporivir (ALV) as monotherapy or combination with ribavirin (RBV) compared to IFN/RBV and ALV/IFN in treatment-naive GT2/3 HCV patients. We describe genotypic and phenotypic changes in patients from this study who experienced a viral breakthrough (VB). METHODS: VITAL-1 study patients on IFN-free regimens that did not achieve RVR (, 25 U/ml) at week 4 were administered IFN/RBV/600 mg QD ALV starting at week 6 (IFN-add-on). VB defined as an increase of HCV RNA .1 log10 above nadir. The HCV NS5A gene was analyzed by population sequencing. The entire NS5A region isolated at baseline and at VB was cloned into a JFH1subgenomic shuttle vector for phenotypic analyses. Selected mutations were engineered into a wild type replicon and their impact on replication fitness and susceptibility to ALV and direct acting antiviral drugs were determined. RESULTS: The VB rate with IFN-free or IFN-add-on regimens was 2.7% (7/ 260). Low ALV drug level (Cmin , 20% of group mean; , 100 ng/mL) was observed (5/ 7 VB patients). Genotypic analysis of the NS5A gene revealed amino acid changes in these patients at the time of VB compared to baseline. Replicons bearing NS5A from patients at breakthrough conferred 1.2-16.9-fold shift over baseline EC50 values; 5/7 had impaired replication fitness. No cross-resistance to other classes of HCV inhibitors was observed. Most frequently identified mutations were D320E (D316E in G2), R347W and A349V in Domain II of NS5A, which by themselves decreased ALV susceptibility by 5-, 4, and 8-fold, respectively, in vitro when engineered into wild-type replicon. Combination of multiple mutations decreased susceptibility further, but generally at the cost of replication fitness. CONCLUSION: In treatment-naive G2/3 patients with viral breakthrough, mutations in NS5A domain II were selected, which individually conferred ≤ 8-fold reduced susceptibility to ALV in vitro. The combination of multiple mutations was required to further reduce susceptibility which is consistent with the high barrier to resistance for host-targeting ALV. Observed genotypic changes did not confer cross-resistance to other classes of HCV inhibitors, supporting the use of ALV in future drug combination therapies.

Sa1031 Mild to Moderate Hepatic Impairment Has No Effect on the Single Dose Pharmacokinetics of Alisporivir (ALV)

BACKGROUND: VITAL-1 study assessed safety, tolerability and antiviral activity of the cyclophilin inhibitor alisporivir (ALV) as monotherapy or combination with ribavirin (RBV) compared to IFN/RBV and ALV/IFN in treatment-naive GT2/3 HCV patients. We describe genotypic and phenotypic changes in patients from this study who experienced a viral breakthrough (VB). METHODS: VITAL-1 study patients on IFN-free regimens that did not achieve RVR (, 25 U/ml) at week 4 were administered IFN/RBV/600 mg QD ALV starting at week 6 (IFN-add-on). VB defined as an increase of HCV RNA .1 log10 above nadir. The HCV NS5A gene was analyzed by population sequencing. The entire NS5A region isolated at baseline and at VB was cloned into a JFH1subgenomic shuttle vector for phenotypic analyses. Selected mutations were engineered into a wild type replicon and their impact on replication fitness and susceptibility to ALV and direct acting antiviral drugs were determined. RESULTS: The VB rate with IFN-free or IFN-add-on regimens was 2.7% (7/ 260). Low ALV drug level (Cmin , 20% of group mean; , 100 ng/mL) was observed (5/ 7 VB patients). Genotypic analysis of the NS5A gene revealed amino acid changes in these patients at the time of VB compared to baseline. Replicons bearing NS5A from patients at breakthrough conferred 1.2-16.9-fold shift over baseline EC50 values; 5/7 had impaired replication fitness. No cross-resistance to other classes of HCV inhibitors was observed. Most frequently identified mutations were D320E (D316E in G2), R347W and A349V in Domain II of NS5A, which by themselves decreased ALV susceptibility by 5-, 4, and 8-fold, respectively, in vitro when engineered into wild-type replicon. Combination of multiple mutations decreased susceptibility further, but generally at the cost of replication fitness. CONCLUSION: In treatment-naive G2/3 patients with viral breakthrough, mutations in NS5A domain II were selected, which individually conferred ≤ 8-fold reduced susceptibility to ALV in vitro. The combination of multiple mutations was required to further reduce susceptibility which is consistent with the high barrier to resistance for host-targeting ALV. Observed genotypic changes did not confer cross-resistance to other classes of HCV inhibitors, supporting the use of ALV in future drug combination therapies.

Sa1030 Alisporivir Interferon-Free G2/3 Vital-1 Study: Low Rate of Viral Breakthrough and Combination of Multiple NS5A Domain II Mutations Required to Reduce Susceptibility to Alisporivir In Vitro

BACKGROUND: VITAL-1 study assessed safety, tolerability and antiviral activity of the cyclophilin inhibitor alisporivir (ALV) as monotherapy or combination with ribavirin (RBV) compared to IFN/RBV and ALV/IFN in treatment-naive GT2/3 HCV patients. We describe genotypic and phenotypic changes in patients from this study who experienced a viral breakthrough (VB). METHODS: VITAL-1 study patients on IFN-free regimens that did not achieve RVR (, 25 U/ml) at week 4 were administered IFN/RBV/600 mg QD ALV starting at week 6 (IFN-add-on). VB defined as an increase of HCV RNA .1 log10 above nadir. The HCV NS5A gene was analyzed by population sequencing. The entire NS5A region isolated at baseline and at VB was cloned into a JFH1subgenomic shuttle vector for phenotypic analyses. Selected mutations were engineered into a wild type replicon and their impact on replication fitness and susceptibility to ALV and direct acting antiviral drugs were determined. RESULTS: The VB rate with IFN-free or IFN-add-on regimens was 2.7% (7/ 260). Low ALV drug level (Cmin , 20% of group mean; , 100 ng/mL) was observed (5/ 7 VB patients). Genotypic analysis of the NS5A gene revealed amino acid changes in these patients at the time of VB compared to baseline. Replicons bearing NS5A from patients at breakthrough conferred 1.2-16.9-fold shift over baseline EC50 values; 5/7 had impaired replication fitness. No cross-resistance to other classes of HCV inhibitors was observed. Most frequently identified mutations were D320E (D316E in G2), R347W and A349V in Domain II of NS5A, which by themselves decreased ALV susceptibility by 5-, 4, and 8-fold, respectively, in vitro when engineered into wild-type replicon. Combination of multiple mutations decreased susceptibility further, but generally at the cost of replication fitness. CONCLUSION: In treatment-naive G2/3 patients with viral breakthrough, mutations in NS5A domain II were selected, which individually conferred ≤ 8-fold reduced susceptibility to ALV in vitro. The combination of multiple mutations was required to further reduce susceptibility which is consistent with the high barrier to resistance for host-targeting ALV. Observed genotypic changes did not confer cross-resistance to other classes of HCV inhibitors, supporting the use of ALV in future drug combination therapies.

Multiple Mutations in Hepatitis C Virus NS5A Domain II Are Required To Confer a Significant Level of Resistance to Alisporivir

Alisporivir is the most advanced host-targeting antiviral cyclophilin (Cyp) inhibitor in phase III studies and has demonstrated a great deal of promise in decreasing hepatitis C virus (HCV) viremia in infected patients. In an attempt to further elucidate the mechanism of action of alisporivir, HCV replicons resistant to the drug were selected. Interestingly, mutations constantly arose in domain II of NS5A. To demonstrate that these mutations are responsible for drug resistance, they were reintroduced into the parental HCV genome, and the resulting mutant viruses were tested for replication in the presence of alisporivir or in the absence of the alisporivir target, CypA. We also examined the effect of the mutations on NS5A binding to itself (oligomerization), CypA, RNA, and NS5B. Importantly, the mutations did not affect any of these interactions. Moreover, the mutations did not preserve NS5A-CypA interactions from alisporivir rupture. NS5A mutations alone render HCV only slightly resistant to alisporivir. In sharp contrast, when multiple NS5A mutations are combined, significant resistance was observed. The introduction of multiple mutations in NS5A significantly restored viral replication in CypA knockdown cells. Interestingly, the combination of NS5A mutations renders HCV resistant to all classes of Cyp inhibitors. This study suggests that a combination of multiple mutations in domain II of NS5A rather than a single mutation is required to render HCV significantly and universally resistant to Cyp inhibitors. This in accordance with in vivo data that suggest that alisporivir is associated with a low potential for development of viral resistance.

Extensive Ace2 duplication and multiple mutations on Ace1 and Ace2 are related with high level of organophosphates resistance in Aphis gossypii

Aphis gossypii (Glover) has been found to possess multiple mutations in the acetylcholinesterase (AChE) gene (Ace) that might involve target site insensitivity. In vitro functional expression of AChEs reveals that the resistant Ace1 (Ace1R) and Ace2 (Ace2R) were significantly less inhibited by eserine, omethoate, and malaoxon than the susceptible Ace1 (Ace1S) and Ace2 (Ace2S). Furthermore, in both the mutant and susceptible AChEs, Ace2 was significantly less sensitive to eserine, omethoate, and malaoxon than Ace1. These results suggested that both the mutant Ace1 and Ace2 were responsible for omethoate resistance, while the mutant Ace2 played a major role in insecticide resistance. The DNA copy number and transcription level of Ace2 were 1.52- and 1.88-fold higher in the ORR strain than in the OSS strain. Furthermore, the DNA copy number and transcription level of Ace2 were significantly higher than that of Ace1 in either OSS or ORR strains, demonstrating the involvement of Ace2 gene duplication in resistance. Thus, the authors conclude that omethoate resistance in cotton aphids appears to have evolved through a combination of multiple mutations and extensive Ace2R gene duplication.

Mutagenesis of pairwise combinations of histone amino-terminal tails reveals functional redundancy in budding yeast

A large body of literature provides compelling evidence for the role of evolutionarily conserved core histone residues in various biological processes. However, site-directed mutagenesis of individual residues that are known to be sites of posttranslational modifications often does not result in clear phenotypic defects. In some cases, the combination of multiple mutations can give rise to stronger phenotypes, implying functional redundancy between distinct residues on histones. Here, we examined the "histone redundancy hypothesis" by characterizing double deletion of all pairwise combinations of amino-terminal tails (N-tails) from the four core histones encoded in budding yeast. First, we found that multiple lysine residues on the N-tails of both H2A and H4 are redundantly involved in cell viability. Second, simultaneous deletion of N-tails from H2A and H3 leads to a severe growth defect, which is correlated with perturbed gross chromatin structure in the mutant cells. Finally, by combining point mutations on H3 with deletion of the H2A N-tail, we revealed a redundant role for lysine 4 on H3 and the H2A N-tail in hydroxyurea-mediated response. Altogether, these data suggest that the N-tails of core histones share previously unrecognized, potentially redundant functions that, in some cases are different from those of the widely accepted H2A/H2B and H3/H4 dimer pairs.

HIV-1 Protease with 20 Mutations Exhibits Extreme Resistance to Clinical Inhibitors through Coordinated Structural Rearrangements

The escape mutant of HIV-1 protease (PR) containing 20 mutations (PR20) undergoes efficient polyprotein processing even in the presence of clinical protease inhibitors (PIs). PR20 shows >3 orders of magnitude decreased affinity for PIs darunavir (DRV) and saquinavir (SQV) relative to PR. Crystal structures of PR20 crystallized with yttrium, substrate analogue p2-NC, DRV, and SQV reveal three distinct conformations of the flexible flaps and diminished interactions with inhibitors through the combination of multiple mutations. PR20 with yttrium at the active site exhibits widely separated flaps lacking the usual intersubunit contacts seen in other inhibitor-free dimers. Mutations of residues 35-37 in the hinge loop eliminate interactions and perturb the flap conformation. Crystals of PR20/p2-NC contain one uninhibited dimer with one very open flap and one closed flap and a second inhibitor-bound dimer in the closed form showing six fewer hydrogen bonds with the substrate analogue relative to wild-type PR. PR20 complexes with PIs exhibit expanded S2/S2' pockets and fewer PI interactions arising from coordinated effects of mutations throughout the structure, in agreement with the strikingly reduced affinity. In particular, insertion of the large aromatic side chains of L10F and L33F alters intersubunit interactions and widens the PI binding site through a network of hydrophobic contacts. The two very open conformations of PR20 as well as the expanded binding site of the inhibitor-bound closed form suggest possible approaches for modifying inhibitors to target extreme drug-resistant HIV.

Distinguishing HIV-1 drug resistance, accessory, and viral fitness mutations using conditional selection pressure analysis of treated versus untreated patient samples

BackgroundHIV can evolve drug resistance rapidly in response to new drug treatments, often through a combination of multiple mutations [1-3]. It would be useful to develop automated analyses of HIV sequence polymorphism that are able to predict drug resistance mutations, and to distinguish different types of functional roles among such mutations, for example, those that directly cause drug resistance, versus those that play an accessory role. Detecting functional interactions between mutations is essential for this classification. We have adapted a well-known measure of evolutionary selection pressure (Ka/Ks) and developed a conditional Ka/Ks approach to detect important interactions.ResultsWe have applied this analysis to four independent HIV protease sequencing datasets: 50,000 clinical samples sequenced by Specialty Laboratories, Inc.; 1800 samples from patients treated with protease inhibitors; 2600 samples from untreated patients; 400 samples from untreated African patients. We have identified 428 mutation interactions in Specialty dataset with statistical significance and we were able to distinguish primary vs. accessory mutations for many well-studied examples. Amino acid interactions identified by conditional Ka/Ks matched 80 of 92 pair wise interactions found by a completely independent study of HIV protease (p-value for this match is significant: 10-70). Furthermore, Ka/Ks selection pressure results were highly reproducible among these independent datasets, both qualitatively and quantitatively, suggesting that they are detecting real drug-resistance and viral fitness mutations in the wild HIV-1 population.ConclusionConditional Ka/Ks analysis can detect mutation interactions and distinguish primary vs. accessory mutations in HIV-1. Ka/Ks analysis of treated vs. untreated patient data can distinguish drug-resistance vs. viral fitness mutations. Verification of these results would require longitudinal studies. The result provides a valuable resource for AIDS research and will be available for open access upon publication at ReviewersThis article was reviewed by Wen-Hsiung Li (nominated by Eugene V. Koonin), Robert Shafer (nominated by Eugene V. Koonin), and Shamil Sunyaev.

생육조건에 따른 Salmonella typhimurium의 숙주세포 부착성

Salmonella typhimurium TML이 Int-407 숙주세포에 부착하는 정도는 S. typhimurium이 낮은 농도의 철이 함유된 배지에서 배양되었을 때보다 LB 액체배지 또는 높은 농도의 철이 함유된 배지에서 생육한 것에서 약 10배 정도의 높은 수준으로 관찰되었다. 고농도의 철이 포함된 배양조건이 살모넬라가 숙주세포에 부착시키는 정도를 향상시키는데 반해, 칼슘, 코발트, 구리, 인산, 마그네슘 그리고 망간과 같은 다른 양이온은 그렇지 않다. 이것은 아마도 철이 Salmonella가 부착에 필요한 요소들의 발현을 활성화하는 역할을 하는 것으로 추정된다. 철 농도에 따른 부착정도의 차이들은 type 1 fimbriae, mannose resistant hemagglutinin과 flagellum등을 생성하지 않는 다양한 S. typhimurium 돌연변이주들에서도 관찰되는 것으로 미루어보아 이들 구조체들과 상관이 없는 밝혀지지 않은 어떤 인자가 부착성 증가에 관여하는 것으로 사료된다. fur유전자가 불활성화된 S. typhimurium 돌연변이주의 부착성이 야생형 Salmonella와 유사한 방식으로 철에 의해 조절되었는데, 이는 철 농도에 따른 부착성의 변화에 관여하는 잠재적인자의 발현이 Fur 단백질과 독립적으로 이루어진다는 것을 나타낸다. An approximately 10-fold higher level of adherence of Salmonella typhimurium strain TML to Int-407 cells was observed with organisms grown in Luria broth or in high-iron containing medium than those grown in low-iron containing medium. Iron specifically enhanced adherence, while other cations such as calcium, cobalt, copper, potassium, magnesium and manganese did not. It was suggested that iron did not act as a passive ligand - probably it stimulated production of bacterial factors necessary for adherence. A similar pattern of iron modulation of adhesiveness was also seen in Salmonella mutants with single or different combinations of multiple mutations in genes encoding the mannose sensitive hemagglutinin (type 1 fimbriae), mannose resistant hemagglutinin and flagellum. The adhesiveness of an isogenic fur mutant was modulated by iron in a manner similar to the wild-type strain, suggesting that iron modulation of adherence is independent of the fur gene product.

Genetic analysis of the ADGF multigene family by homologous recombination and gene conversion in Drosophila.

Many Drosophila genes exist as members of multigene families and within each family the members can be functionally redundant, making it difficult to identify them by classical mutagenesis techniques based on phenotypic screening. We have addressed this problem in a genetic analysis of a novel family of six adenosine deaminase-related growth factors (ADGFs). We used ends-in targeting to introduce mutations into five of the six ADGF genes, taking advantage of the fact that five of the family members are encoded by a three-gene cluster and a two-gene cluster. We used two targeting constructs to introduce loss-of-function mutations into all five genes, as well as to isolate different combinations of multiple mutations, independent of phenotypic consequences. The results show that (1) it is possible to use ends-in targeting to disrupt gene clusters; (2) gene conversion, which is usually considered a complication in gene targeting, can be used to help recover different mutant combinations in a single screening procedure; (3) the reduction of duplication to a single copy by induction of a double-strand break is better explained by the single-strand annealing mechanism than by simple crossing over between repeats; and (4) loss of function of the most abundantly expressed family member (ADGF-A) leads to disintegration of the fat body and the development of melanotic tumors in mutant larvae.

CK2 Phosphorylation of the Armadillo Repeat Region of β-Catenin Potentiates Wnt Signaling

Protein kinase CK2 is a ubiquitous serine/threonine kinase involved in many biological processes. It is overexpressed in many malignancies including rodent and human breast cancer, and is up-regulated in Wnt-transfected mammary epithelial cells, where it can be found in a complex with dishevelled and beta-catenin. beta-Catenin is a substrate for CK2 and inhibition of CK2 reduces levels of beta-catenin and dishevelled. Here we report that inhibition of CK2 using pharmacologic agents or expression of kinase inactive subunits reduces beta-catenin-dependent transcription and protein levels in a proteasome-dependent fashion. The major region of phosphorylation of beta-catenin by CK2 is the central armadillo repeat domain, where carrier proteins like axin and the adenomatous polyposis coli gene product APC interact with beta-catenin. The major CK2 phosphorylation site in this domain is Thr393, a solvent-accessible residue in a key hinge region of the molecule. Mutation of this single amino acid reduces beta-catenin phosphorylation, cotranscriptional activity, and stability. Thus, CK2 is a positive regulator of Wnt signaling through phosphorylation of beta-catenin at Thr393, leading to proteasome resistance and increased protein and co-transcriptional activity.

Selection and characterization of beta-lactam-beta-lactamase inactivator-resistant mutants following PCR mutagenesis of the TEM-1 beta-lactamase gene.

Mechanism-based inactivators of beta-lactamases are used to overcome the resistance of clinical pathogens to beta-lactam antibiotics. This strategy can itself be overcome by mutations of the beta-lactamase that compromise the effectiveness of their inactivation. We used PCR mutagenesis of the TEM-1 beta-lactamase gene and sequenced the genes of 20 mutants that grew in the presence of ampicillin-clavulanate. Eleven different mutant genes from these strains contained from 1 to 10 mutations. Each had a replacement of one of the four residues, Met69, Ser130, Arg244, and Asn276, whose substitutions by themselves had been shown to result in inhibitor resistance. None of the mutant enzymes with multiple amino acid substitutions generated in this study conferred higher levels of resistance to ampicillin alone or ampicillin with beta-lactamase inactivators (clavulanate, sulbactam, or tazobactam) than the levels of resistance conferred by the corresponding single-mutant enzymes. Of the four enzymes with just a single mutation (Ser130Gly, Arg244Cys, Arg244Ser, or Asn276Asp), the Asn276Asp beta-lactamase conferred a wild-type level of ampicillin resistance and the highest levels of resistance to ampicillin in the presence of inhibitors. Site-directed random mutagenesis of the Ser130 codon yielded no other mutant with replacement of Ser130 besides Ser130Gly that produced ampicillin-clavulanate resistance. Thus, despite PCR mutagenesis we found no new mutant TEM beta-lactamase that conferred a level of resistance to ampicillin plus inactivators greater than that produced by the single-mutation enzymes that have already been reported in clinical isolates. Although this is reassuring, one must caution that other combinations of multiple mutations might still produce unexpected resistance.