Resistance Gene Sequences Research Articles

DNA and All-Trans Retinoic Acid as Immunotherapy or Add-on Adjuvants to 5-Azacytidine In Myelodysplastic Syndromes

AbstractAbstract 2938 Background:Myelodysplastic syndromes (MDS) are clonal stem cell hematological disorders characterized by ineffective hematopoiesis leading to cytopenia, which evolve to acute myeloid leukemia (AML). The ability of the DNA vaccination to induce effective immune responses has been demonstrated in different preclinical models of diseases. Having previously shown that DNA vaccination with a PMLRARaFrC plasmid in combination with all-trans retinoic acid (ATRA) induced long term remissions with appropriate immune responses in a mouse model of acute promyelocytic leukemia (APL), our aim was to use this strategy in a mouse model of MDS we have created using transgenic mice bearing a mutant NRAS and overexpression of human BCL-2. Methods:We used our MDS-like triple transgenic mice bearing NRASD12/MMTVLTRtTA/TetoBCL-2 (Omidvar et al Cancer Res 67:11657-67, 2007). We cloned part of the kanamycin resistance gene and adjacent pVax1 plasmid sequences flipped so the antisense sequences were inserted into the pVax1 vector (named Flipper). These sequences code for 5 polypeptides. We treated the MDS mice with either DNA alone, ATRA alone (10mg daily release for 21 days) or a combination of ATRA+DNA or as add-on therapy with 5-azacytidine (5-aza). 5-aza (5mg/kg) was administered 3 times a week and continued until death. Treatment with ATRA and/or DNA was initiated after 12 courses of 5-aza injections. Results and Conclusions:Sequencing of the Flipper insert predicted 5 peptides, which were synthesized and shown not to affect growth in vitro of myeloid leukemic NB4, K562 and p39 cell lines. In vivo injections of healthy mice with these peptides had no effect on mortality. The inserted sequences did not appear to have detrimental effects. The studies on our MDS mouse model show that DNA vaccination, alone or in combination with ATRA induces a long-term survival of the treated mice compared to untreated controls (p<0,0001). DNA vaccination results in an increase of interferon-gamma secretion as measured by ELISPOT, increased CD4+/CD44hi/CD62-Llo memory T cells in the peripheral blood and maintains stable disease as determined by the persistence of low peripheral blood platelet counts and the of the Mac-1hi/GR-1lo populations, which mark the primitive cells and the ERK phosphorylation signatures measured by the NanoPro (Cell Biosciences). Memory T-cell levels increased 3-fold (at 6% compared to 2% in healthy controls) and remained high. The DNA vaccine stabilized the disease and its protective effect may persist for up to 10 months with increased expression of MYD88, which is downstream of toll-like receptors, in long-term survivors, suggesting the activation of this DNA pathway. ATRA alone also had efficacy in this model. As there was no evidence of differentiation as the Mac-1hi/Gr-1lo blast cell population remained unchanged after treatment compared to the levels before treatment, it is possible that in this setting where the leukemic stem cells are pro-apoptotic, that ATRA is acting as an immuno-adjuvant and the antigen shedding that occurs allows cross presentation and induces immune responses potentiated by ATRA. However, the disease in this group progresses and the mice die earlier than the other treatment groups (mean survival of 4.5 months compared with the untreated group of 1 month, p=0.0016). Vaccination with an adjuvant DNA alone or in combination with ATRA, results in a significant extension of lifespan in MDS mice. As add-on therapy to 5-aza the response rate increases from 20% with 5-aza alone to 50% with either ATRA or DNA and 100% with the 5-aza, ATRA and DNA combination. These studies suggest that adjuvant therapy with ATRA+DNA may have a role in addition to conventional therapy in prolonging remissions and may be promising for clinical trials. Disclosures:Fenaux:Novartis, Janssen, Cilag, Roche, Amgem, GSK, Merck and Cephalon: Honoraria, Research Funding. Chomienne:Vivavacs SAS: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Padua:Vivavacs SAS: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Developing Specific Markers and Improving Genetic Mapping for a Major Locus &lt;em&gt;Tyr1&lt;/em&gt; of Citrus Nematode Resistance

The NBS-LRR class resistance-gene candidate sequences (Pt8a and Pt9a) were used to develop new specific markers to the citrus nematode resistance gene locus Tyr1 . By high-density colony screening, over 200 positive clones were pulled out from the BAC library. A few of the clones were found to be closely linked with the Tyr1 region, because the primers from these clones insert sequence produced polymorphism which matched up with the phenotype after bulked segregant analysis. By primer walking approach, three integrate NBS-LRR class resistance-gene sequences were tagged and identified separately in three clones (7A4, 4L17 and 29F20). More specific markers were developed from these tagged sequences and relatively high-density genetic maps were constructed by incorporating the newly developed markers and previously developed markers in the ‘9145 family’. New markers were applied in ‘9401 family’ trying to estimate roughly the genetic distance between the Ctv and Tyr1 region.

The effective expression of xylanase gene in Candida utilis by 18S rDNA targeted homologous recombination in pGLR9K

In order to test whether 18S rDNA can influence positively xylanase gene effective expression in the yeast of Candida utilis, a targeting vector pGLR9K-XA was constructed by adding an interested gene xynA from Streptomyces olivaceoviridis into the vector pGLR9K which is constructed by ourselves. pGLR9K contains the 18S rDNA, GAP promoter and CYH resistance gene sequence, all of which is from C. utilis. Then the vector pGLR9K-XA was transformed into C. utilis. To test the vector and transformed system, PCR, Southern blot and DNS methods were used. The results showed that xylanase gene can be detected in the chromosome DNA of recombinant C. utilis and the enzyme activity of xylanase is up to 60 IU ml(-1) in the study. It is suggested that this system can be used to express exogenous genes in C. utilis as a bioreactors. This is the first report that xylanase gene was expressed in C. utilis.

Performance Assessment of the GenoType MTBDR plus Test and DNA Sequencing in Detection of Multidrug-Resistant Mycobacterium tuberculosis

To facilitate the management of multidrug-resistant (MDR) tuberculosis, two nucleic acid sequence-based methods, the GenoType MTBDRplus test and DNA sequencing, were assessed for the rapid detection of drug-resistant Mycobacterium tuberculosis for the first time in the Asia-Pacific region. The performances of these two assays in detecting the presence of rifampin (rifampicin) (RIF) and isoniazid (INH) resistance-associated mutations in the rpoB, katG, inhA regulatory region, inhA, and oxyR-ahpC genes were compared to that of a conventional agar proportion drug susceptibility test. A total of 242 MDR and 30 pansusceptible M. tuberculosis isolates were evaluated in this study. The sensitivities obtained for RIF-resistant detection by the GenoType MTBDRplus test and by resistance gene sequencing were 95.5% and 97.9%, respectively. The sensitivities for INH resistance detection by the GenoType MTBDRplus test and by resistance gene sequencing were 81.8% and 93.4%, respectively. Together, the sensitivity for MDR tuberculosis detection was 78.5% with the GenoType MTBDRplus test and 91.3% by resistance gene sequencing. The specificity for RIF resistance, INH resistance, and MDR detection was 100% by both methods. The GenoType MTBDRplus test has the advantage of a short turnaround time for drug-resistant M. tuberculosis detection. Overall, the two assays performed equally well in detecting RIF resistance (P = 0.13). However, DNA sequencing demonstrated superior performance in detecting INH resistance (P < 0.001) and MDR tuberculosis (P < 0.001). We suggest that new alleles of INH resistance genes should be evaluated to improve the sensitivity of the GenoType MTBDRplus test, especially for different geographic areas with genetically diverse M. tuberculosis strains.

Multi‐drug resistance (MDR1) gene identification and sequence in horses

P‐glycoprotein (P‐gp), a membrane transporter encoded in the multi‐drug resistance (MDR1) gene, has been identified in many species but not in equine. P‐gp interferes with bioavailability and disposition of many drugs and may explain adverse drug‐induced effects after intravenous administration and poor response with oral administration.The hypothesis is that the MDR1 gene is present in horses. We identified the presence of the equine MDR1 gene, obtained the gene sequence and detected its presence in normal equine tissues.To identify the equine MDR1 gene, total RNA was extracted from intestinal samples, was reverse transcribed to cDNA and used as a template for PCR. Primers were designed by MacVector 65.3 software using the Gen Bank AY360144 MDR1 cDNA rabbit sequence. Real Time TaqMan RT‐PCR was used to detect the presence of MDR1 gene in the intestine, brain, and spinal cord from six horses.Our sequence was compared to the MDR1 rabbit sequence and the horse partial MDR1 sequence demonstrated high similarity with this (84%) and other species. Our RT‐PCR data confirmed the presence of the MDR1 gene in the intestine, brain, and spinal cord of all 6 horses.This data may improve pharmacokinetic and pharmacodynamic studies since the presence of this gene may predispose horses to differences in enteric absorption of some therapeutic agents.Funding provided by the LSU Equine Health Studies Program.

Resistance Gene Analogs (RGA) as a tool in fruit tree's breeding

Breeding for pest and disease resistance comes as a major objective behind the fruit traits. To increase the effectiveness of fruit resistance breeding application of the Marker Assisted Selection ( MAS) is advantageous. For generating molecular markers which enable the following of interesting traits basically two methods are available: targeted marker design based on conservative region of already known Resistance ( R) gene sequences or randomly generated markers. The creation and the application of these homology based markers are the object of this review in the main temperate zone fruit species.

Expansion and countrywide dissemination of ST11, ST15 and ST147 ciprofloxacin-resistant CTX-M-15-type -lactamase-producing Klebsiella pneumoniae epidemic clones in Hungary in 2005--the new 'MRSAs'?

To investigate the molecular epidemiology of ciprofloxacin-resistant CTX-M-15-producing Klebsiella pneumoniae epidemic clones (ECs) isolated from six nosocomial outbreaks and sporadic cases during 2005 in Hungary. Two hundred and eighty-one extended-spectrum beta-lactamase (ESBL)-producing K. pneumoniae clinical isolates collected from 41 centres were submitted to the National ESBL Reference Laboratory for further investigations. Of the 281 strains, 75 isolates proved to be SHV producers, whereas 6 isolates were ciprofloxacin-susceptible CTX-M-type ESBL producers. One hundred and ninety-six ciprofloxacin-resistant CTX-M-type beta-lactamase-producing isolates collected from 35 centres were subjected to macrorestriction profile analysis. Furthermore, molecular typing was performed by PCR and sequencing of several antibiotic resistance genes, plasmid profile analysis, transfer of resistance determinants and multilocus sequence typing (MLST). PFGE revealed the existence of three genetic clusters defined as ECs, where 129 isolates belonged to the previously described Hungarian EC (HEC), 46 isolates to epidemic clone II (EC II) and 21 isolates to epidemic clone III (EC III), respectively. All isolates harboured plasmids ranging from 2.0 to 230 kb. PstI digestion of plasmid DNA from transconjugants/transformants revealed diverse restriction patterns from distinct ECs. Sequence analysis of beta-lactamase genes from 19 selected isolates detected bla(CTX-M-15) and bla(OXA-1) in strains from all three ECs and bla(TEM-1) in EC III isolates located on large plasmids. ISEcpI associated with CTX-M-15 was detected only on a 50 kb non-conjugative plasmid from EC III. MLST identified three allelic profiles: ST 15 (HEC), ST 11 (EC III) and the novel ST 147 (EC II), which correspond to the PFGE clusters, respectively. In 2005, 97% of all CTX-M-producing K. pneumoniae isolates detected across Hungary were highly ciprofloxacin-resistant CTX-M-15 producers and represented just three stable genetic clones.

Contributions of domesticated plant studies to our understanding of plant evolution.

Plant evolutionary theory has been greatly enriched by studies on crop species. Over the last century, important information has been generated on many aspects of population biology, speciation and polyploid genetics. Searches for quantitative trait loci (QTL) in crop species have uncovered numerous blocks of genes that have dramatic effects on adaptation, particularly during the domestication process. Many of these QTL have epistatic and pleiotropic effects making rapid evolutionary change possible. Most of the pioneering work on the molecular basis of self-incompatibility has been conducted on crop species, along with the sequencing of the phytopathogenic resistance genes (R genes) responsible for the 'gene-to-gene' relations of coevolution observed in host-pathogen relationships. Some of the better examples of co-adaptation and early acting inbreeding depression have also been elucidated in crops. Crop-wild progenitor interactions have provided rich opportunities to study the evolution of novel adaptations subsequent to hybridization. Most crop/wild F1 hybrids have reduced fitness, but in some instances the crop relatives have acquired genes that make them more efficient weeds through crop mimicry. Studies on autopolyploid alfalfa and potato have uncovered the means by which polyploid gametes are formed and have led to hypotheses about how multiallelic interactions are associated with fitness and self-fertility. Research on the cole crops and wheat has discovered that newly formed polyploids can undergo dramatic genome rearrangements that could lead to rapid evolutionary change. Many more important evolutionary discoveries are on the horizon, now that the whole genome sequence is available of the two major subspecies of rice Oryza sativa ssp. japonica and O. sativa ssp. indica. The rice sequence data can be used to study the origin of genes and gene families, track rates of sequence divergence over time, and provide hints about how genes evolve and generate products with novel biological properties. The rice sequence data has already been mined to show that transposable elements often carry fragments of cellular genes. This type of genome shuffling could play a role in creating novel, reorganized genes with new adaptive properties.

Prevalence of Antimicrobial Resistance Genes inListeria monocytogenesIsolated from Dairy Farms

Antimicrobial resistance of Listeria monocytogenes (n = 38) isolated from the four dairy farms to 15 antimicrobial agents was evaluated. All 38 L. monocytogenes isolates from the four farms evaluated were resistant to more than one antimicrobial in different combinations. All L. monocytogenes isolates evaluated were resistant to cephalosporin C (minimum inhibitory concentration [MIC] > or = 512 microg/mL), streptomycin (MIC > or = 32) and trimethoprim (MIC > or = 512). Most L. monocytogenes isolates were resistant to ampicillin (92%, MIC > or = 2), rifampicin (84%, MIC > or = 4), rifamycin (84%, MIC > or = 4), and florfenicol (66%, MIC > or = 32) and some were resistant to tetracycline (45%, MIC > or = 16), penicillin G (40%, MIC > or = 2) and chloramphenicol (32%, MIC > or = 32). All L. monocytogenes isolates were susceptible to amoxicillin, erythromycin, gentamicin, kanamycin and vancomycin. Susceptibility of L. monocytogenes to the antimicrobials evaluated was quite consistent among the dairy farms evaluated. However, some variability in antimicrobial susceptibility among dairy farms was noted. Nineteen of 38 L. monocytogenes isolates contained more than one antimicrobial resistance gene sequence. A high frequency of floR (66%) was found in L. monocytogenes followed by penA (37%), strA (34%), tetA (32%), and sulI (16%). Other tetracycline resistance genes (tetB, tetC, tetD, tetE, and tetG) and other antimicrobial resistance genes (cmlA, strB, aadA, sulI, vanA, vanB, ampC, ermB, ereA, and ereB) were not found in any of the L. monocytogenes isolates from the four dairy farms. Results of the present study demonstrated that L. monocytogenes isolated from the dairy farm environment were resistant to many antimicrobials and contained one or more antimicrobial resistance genes.

Identification of a large cluster of coiled coil-nucleotide binding site–leucine rich repeat-type genes from the Rps1 region containing Phytophthora resistance genes in soybean

Fifteen Rps genes confer resistance against the oomycete pathogen Phytophthora sojae, which causes root and stem rot disease in soybean. We have isolated a disease resistance gene-like sequence from the genomic region containing Rps1-k. Four classes of cDNA of the sequence were isolated from etiolated hypocotyl tissues that express the Rps1-k-encoded Phytophthora resistance. Sequence analyses of a cDNA clone showed that the sequence is a member of the coiled coil-nucleotide binding site-leucine rich repeat (CC-NBS-LRR)-type of disease resistance genes. It showed 36% identity to the recently cloned soybean resistance gene Rpg1-b, which confers resistance against Pseudomonas syringae pv. glycinea, and 56% and 38% sequence identity to putative resistance gene sequences from lotus and Medicago truncatula, respectively. The soybean genome contains about 38 copies of the sequence. Most of these copies are clustered in approximately 600 kb of contiguous DNA of the Rps1-k region. We have identified a recombinant that carries both rps1-k- and Rps1-k-haplotype-specific allelomorphs of two Rps1-k-linked molecular markers. An unequal crossover event presumably led to duplication of alleles for these two physically linked molecular markers. We hypothesize that the unequal crossing over was one of the mechanisms involved in tandem duplication of CC-NBS-LRR sequences in the Rps1-k region.

Antibiotic Resistance Genes Online (ARGO): A Database on vancomycin and b-lactam resistance genes

Vancomycin and beta-lactams are antibiotics that inhibit gram positive bacteria by interfering with cell wall synthesis. However, continuous use of antibiotics results in the emergence of multi-drug resistant (MDR) bacterial strains. Here, we describe ARGO, a database containing gene sequences conferring resistance to these two classes of antibiotics. It is designed as a resource to enhance research on the prevalence and spread of antibiotic resistance genes. ARGO is the first attempt to compile the resistance gene sequence data with state specific information. AGRO is available for free at http://www.argodb.org/

The introgressed segment carrying rust resistance genes Yr17, Lr37 and Sr38 in wheat can be assayed by a cloned disease resistance gene-like sequence

A cloned gene sequence (Vrga1D), with features of the nucleotide-binding-site leucine-rich repeat class of disease resistance (R) gene sequence super family, was previously shown to belong to a family of five gene members derived from a Triticum ventricosum Ces. (syn. Aegilops ventricosa Tausch) segment in wheat (Triticum aestivum L.). This gene family was introgressed, together with the linked rust resistance genes Yr17, Lr37 and Sr38 from T. ventricosum, to wheat chromosome 2AS. An independently derived T. ventricosum segment carrying a leaf rust resistance gene in a French wheat cultivar, was shown to exhibit a rust resistance response equivalent to Lr37 as well as Yr17 and Sr38. DNA probes from different regions of the Vrga1D clone consistently detected the presence of RFLPs associated with the introgressed segment carrying the resistance genes Yr17, Lr37 and Sr38 present in diverse wheat genotypes from Australia, Canada, France and the UK. Our results showed that the transfer of the T. ventricosum- derived Vrga1 gene members and the rust resistance genes were always accompanied by the loss of a corresponding set of Vrga1-related gene members in recipient wheat cultivars presumed to be of homoeoallelic origin. A PCR assay, based on sequences from the 3"-untranslated region of a Vrga1 gene member isolated from the T. ventricosum donor line of the introgressed segment, was developed. The PCR assay detected the presence of the introgressed rust resistance genes across the diverse wheat backgrounds and should be useful in marker- assisted selection in wheat breeding.

Separation and purification of plasmid mixtures by continuous elution electrophoresis.

Separation and purification of plasmid mixtures by continuous elution electrophoresis.

Cloning and characterization of NBS-LRR class resistance-gene candidate sequences in citrus

Numerous disease resistance gene-like DNA sequences were cloned from an intergeneric hybrid of Poncirus and Citrus, using a PCR approach with degenerate primers designed from conserved NBS (nucleotide-binding site) motifs found in a number of plant resistance genes. Most of the cloned genomic sequences could be translated into polypeptides without stop codons, and the sequences contained the characteristic motifs found in the NBS-LRR class of plant disease resistance genes. Pairwise comparisons of these polypeptide sequences indicated that they shared various degrees of amino-acid identity and could be grouped into ten classes (RGC1–RGC10). When the sequences of each class were compared with known resistance-gene sequences, the percentage of amino-acid identity ranged from 18.6% to 48%. To facilitate genetic mapping of these sequences and to assess their potential linkage relationship with disease resistance genes in Poncirus, we developed CAPS markers by designing specific primers based on the cloned DNA sequences and subsequently identifying restriction enzymes that revealed genetic polymorphisms. Three of the amplified DNA fragment markers (designated as 18P33a, Pt9a, and Pt8a) were associated with the citrus tristeza virus resistance gene (Ctv), and one fragment (Pt8a) was associated with the major gene responsible for the citrus nematode resistance (Tyr1); both genes are from Poncirus and of importance to citrus survival and production. These polymorphic fragments were located on two local genetic linkage maps of the chromosome region from Ctv to Tyr1. These results indicate that resistance-gene candidate sequences amplified with the NBS-derived degenerate primers are valuable sources for developing markers in disease resistance-gene tagging, mapping, and cloning.

Construction of a bacterial artificial chromosome (BAC) library of Lycopersicon esculentum cv. Stevens and its application to physically map the Sw-5 locus

The Sw-5 gene is a dominantly inherited resistance gene in tomato and functional against a number of tospovirus species. The gene has been mapped on chromosome 9, tightly linked to RFLP markers CT220 and SCAR421. To analyse the Sw-5 locus, a BAC genomic library was constructed of tomato cv. Stevens, homozygous for the Sw-5 gene. The library comprised 18 816 clones with an average insert size of 100 kb, corresponding to two genome equivalents. The library was screened by PCR using primers designed for the CT220 and SCAR421 sequences, resulting in a 250 kb contig of known orientation on the long arm of chromosome 9. Using degenerate primers based on homologous sequences in the nucleotide binding site of resistance gene sequences, three discrete PCR fragments obtained from this contig were cloned and sequenced. Analysis of these fragments revealed a high similarity with numerous resistance genes or resistance gene like sequences. The present data indicate that at least three different resistance gene candidate (RGC) sequences are present in the vicinity of marker CT220, supporting the view that a resistance gene family may be responsible for the unusually broad resistance to tospoviruses conferred by the Sw-5 locus.

Characterization of Plasmodium falciparum isolated from the Amazon region of Brazil: evidence for quinine resistance.

The prevalence and severity of drug-resistant malaria is emerging rapidly in the Amazon basin of Brazil. In support of clinical trials using the new antimalarial drug combination of atovaquone and proguanil, we performed in vitro drug sensitivities, molecular characterization of parasite populations using the circumsporozoite protein, merozoite surface antigen-1 (MSA-1), and MSA-2 markers, and an analysis of the Plasmodium falciparum multidrug resistance (pfmdr1) gene sequence and copy number in 26 isolates of P. falciparum obtained in a gold-mining endemic area in Peixoto de Azevedo, Mato Grosso State. All 26 isolates were found to be resistant to chloroquine (50% inhibitory concentration [IC50] = 100-620 nM) and sensitive to mefloquine (IC50 < 23 nM) and halofantrine (IC50 < 6 nM). The isolates also show reduced susceptibility to quinine (IC50 = 48-280 nM). Sequence analysis of the pfmdr1 gene revealed Asn, Phe, Cys, Asp, and Tyr in positions 86, 184, 1034, 1042, and 1246, respectively. These point mutations were similar to that previously described in other Brazilian isolates. Southern blot analysis revealed no amplification of the pfmdr1 gene. These results suggest that three different mechanisms for drug resistance exist for chloroquine, mefloquine, and quinine.

Inactivation of antibiotics and the dissemination of resistance genes.

The emergence of multidrug-resistant bacteria is a phenomenon of concern to the clinician and the pharmaceutical industry, as it is the major cause of failure in the treatment of infectious diseases. The most common mechanism of resistance in pathogenic bacteria to antibiotics of the aminoglycoside, beta-lactam (penicillins and cephalosporins), and chloramphenicol types involves the enzymic inactivation of the antibiotic by hydrolysis or by formation of inactive derivatives. Such resistance determinants most probably were acquired by pathogenic bacteria from a pool of resistance genes in other microbial genera, including antibiotic-producing organisms. The resistance gene sequences were subsequently integrated by site-specific recombination into several classes of naturally occurring gene expression cassettes (typically "integrons") and disseminated within the microbial population by a variety of gene transfer mechanisms. Although bacterial conjugation once was believed to be restricted in host range, it now appears that this mechanism of transfer permits genetic exchange between many different bacterial genera in nature.

Antibiotic preparations contain DNA: a source of drug resistance genes?

Fluorescence measurements and polymerase chain reaction amplification of streptomycete 16S ribosomal DNA sequences were used to show that a number of antibiotic preparations employed for human and animal use are contaminated with chromosomal DNA of the antibiotic-producing organism. The DNA contains identifiable antibiotic resistance gene sequences; the uptake of this DNA by bacteria and its functional incorporation into bacterial replicons would lead to the generation of antibiotic resistance determinants. We propose that the presence of DNA encoding drug resistance in antibiotic preparations has been a factor in the rapid development of multiple antibiotic resistance in bacteria.

Reduced topoisomerase II and elevated α class glutathione S-transferase expression in a multidrug resistant CHO cell line highly cross-resistant to mitomycin C

We have isolated a multidrug-resistant derivative of Chinese hamster ovary CHO-K1 cells by exposure to progressively increasing concentrations of Adriamycin®. This cell line, designated CHO-Adr r, was 27-fold more resistant than the parental line to Adriamycin and showed similar degrees of cross-resistance to several other topoisomerase II (topo II) inhibitors, including mitoxantrone, daunomycin and etoposide. CHO-Adr r cells showed a lower (4-fold) level of cross-resistance to vincristine and colchicine, drugs associated with the multidrug-resistant phenotype. While CHO-Adr r cells showed no enhanced resistance to several mono- and bi-functional alkylating agents or to UV and ionizing radiation, they were greater than 80-fold resistant to mitomycin C (MMC). There was a 5-fold decreased level of daunomycin accumulation in CHO-Adr r cells compared to CHO-K1 cells and this was associated with increased drug efflux. The resistant cells had amplified multidrug resistance gene ( mdr) sequences and overexpressed ( mdr) mRNA. Verapamil was able to completely reverse Adriamycin resistance but reversal of MMC resistance was only partial, with residual 23-fold resistance. CHO-Adr r cells expressed a 4-fold reduced level of topo II protein but overexpressed an α class (basic) glutathione S-transferase (GST). Analysis of cell hybrids showed that while the level of resistance to Adriamycin dropped by a factor of 3 in CHO-K1/CHO-Adr r hybrids compared to CHO-Adr r/CHO-Adr r hybrids, resistance to MMC dropped 10-fold. Thus, CHO-Adr r cells appear to exhibit simultaneously several different drug resistance mechanisms including MDR and GST overexpression, and topo II reduction.

Three short fragments of Rts1 DNA are responsible for the temperature-sensitive growth phenotype (Tsg) of host bacteria

Rts1 is a multiphenotype drug resistance factor, and one of its phenotypes is temperature-sensitive growth (Tsg) of host bacteria. A 3.65-kb fragment from Rts1 DNA was shown to cause the Tsg phenotype in host cells. This tsg fragment was split by a restriction enzyme, HincII, into four fragments. Two of these fragments were called HincII-S (short) and HincII-L (long), respectively. Each of these two fragments conferred the Tsg phenotype, indicating that, in fact, these two independent regions were responsible for the Tsg phenotype. The HincII-S 783-bp and HincII-L 1,479-bp fragments were sequenced. The region in the HincII-S fragment to which the Tsg phenotype was attributed was narrowed to a 146-bp (nucleotides 1 to 146) fragment by various restriction enzyme digestions. Further digestion of the 146-bp fragment with Bal 31 suggested that the 116-bp (nucleotides 9 to 124) fragment is the minimum sequence required for Tsg. On the other hand, in the HincII-L fragment, a fragment of 249 bp (nucleotides 1210 to 1458) and a fragment of 321 bp (nucleotides 1942 to 2262) contained separate temperature-sensitive growth activity. None of three tsg fragments contained open reading frames. The 249-bp fragment had very weak Tsg activity, while the 321-bp fragment had no Tsg activity. On the other hand, when these two fragments were together in the pUC19 vector, they exhibited very strong Tsg activity equivalent to that of the original 1,479-bp fragment. In addition, two of the 249-bp fragments gave similar, strong Tsg activity. The HincII-L 1,479-bp fragment contained an open reading frame for kanamycin resistance which was found between nucleotides 1423 and 2238. This kanamycin resistance gene sequence was different from that of the reported kanamycin resistance gene of Tn903 at 12 positions which were deduced to change seven amino acids.