Specific selection of deoxycytidine kinase mutants with tritiated deoxyadenosine

We have shown previously that a low concentration of tritiated deoxyadenosine, i.e., 1 microCi/ml, selectively kills wild-type S49 murine lymphoma cells. Mutant cells resistant to [3H] deoxyadenosine lacked adenosine kinase completely but retained a significant level of deoxyadenosine phosphorylating activity. To study further the specificity of [3H] deoxyadenosine selection, lymphoma cell clones resistant to 15 microCi/ml [3H] deoxyadenosine have been derived. The resistant line, S49-dA15, is also resistant to high levels of nonradioactive deoxyadenosine and to deoxyguanosine but remains sensitive to thymidine. The thymidine inhibition of the growth of the mutant, in contrast to that of the wild-type cells, cannot be prevented by deoxycytidine. The mutant line lacks deoxycytidine kinase that also phosphorylates deoxyadenosine. In addition, the mutant cells excrete a large amount of deoxycytidine into culture medium, consistent with a failure of salvage of the nucleoside in the absence of an appropriate kinase, i.e., deoxycytidine kinase. In contrast, a deoxycytidine kinase-deficient cell line that was selected with arabinosylcytosine does not excrete deoxycytidine and contains high deoxycytidine deaminase activity. [3H] Deoxyadenosine can be used as a selective agent for specific selection of deoxycytidine kinase-negative mutants.     

A genetic selection for Caenorhabditis elegans synaptic transmission mutants

We have isolated 165 Caenorhabditis elegans mutants, representing 21 genes, that are resistant to inhibitors of cholinesterase (Ric mutants). Since mutations in 20 of the genes appear not to affect acetylcholine reception, we suggest that reduced acetylcholine release contributes to the Ric phenotype of most Ric mutants. Mutations in 15 of the genes lead to defects in a gamma-aminobutyric acid-dependent behavior; these genes are likely to encode proteins with general, rather than cholinergic-specific, roles in synaptic transmission. Ten of the genes have been cloned. Seven encode homologs of proteins that function in the synaptic vesicle cycle: two encode cholinergic-specific proteins, while five encode general presynaptic proteins. Two other Ric genes encode homologs of G-protein signaling molecules. Our assessment of synaptic function in Ric mutants, combined with the homologies of some Ric mutants to presynaptic proteins, suggests that the analysis of Ric genes will continue to yield insights into the regulation and functioning of synapses.     

De novo protein design: fully automated sequence selection

The first fully automated design and experimental validation of a novel sequence for an entire protein is described. A computational design algorithm based on physical chemical potential functions and stereochemical constraints was used to screen a combinatorial library of 1.9 x 10(27) possible amino acid sequences for compatibility with the design target, a betabetaalpha protein motif based on the polypeptide backbone structure of a zinc finger domain. A BLAST search shows that the designed sequence, full sequence design 1 (FSD-1), has very low identity to any known protein sequence. The solution structure of FSD-1 was solved by nuclear magnetic resonance spectroscopy and indicates that FSD-1 forms a compact well-ordered structure, which is in excellent agreement with the design target structure. This result demonstrates that computational methods can perform the immense combinatorial search required for protein design, and it suggests that an unbiased and quantitative algorithm can be used in various structural contexts.     

Cisplatin resistance and regulation of DNA repair in cAMP-dependent protein kinase mutants

Drug resistance in cancer poses a major problem to the success of chemotherapy. Increased resistance to the DNA-damaging chemotherapeutic drug cisplatin may be associated with a variety of factors including decreased drug accumulation, increased intracellular levels of thiols, and increased DNA repair. We have found that mutants of the Chinese hamster ovary (CHO) and the mouse adrenocortical carcinoma Y1 cells harboring a defective regulatory subunit (RI) of the cAMP-dependent protein kinase (PKA) exhibited increased resistance to cisplatin. These mutants are cross-resistant to other DNA-damaging chemotherapeutic agents, including bleomycin and melphalan. In addition, wild-type CHO cells transfected with and overexpressing the yeast phosphodiesterase gene or a dominant mutant Rl alpha subunit gene also displayed similar increased resistance to cisplatin. However, mutants with altered catalytic (C) subunits showed a sensitivity to cisplatin similar to the wild-type cells. Further analysis by gel shift assay using cisplatin-damaged DNA as probes and nuclear extracts derived from the Rl subunit mutants showed increased binding of nuclear factor(s) to the damaged DNA. In addition, a host cell reactivation assay of DNA repair, using a cisplatin-damaged reporter plasmid, detected enhanced capacity for repair of DNA lesions in the PKA mutants. These results suggest that DNA repair may be increased in the PKA mutants. We speculate that functional inactivation of PKA may result in increased DNA repair and the acquisition of resistance to DNA-damaging anticancer drugs in cancer.     

Aberrant cell division and random FtsZ ring positioning in Escherichia coli cpxA* mutants

In Escherichia coli, certain mutations in the cpxA gene (encoding a sensor kinase of a two-component signal transduction system) randomize the location of FtsZ ring assembly and dramatically affect cell division. However, deletion of the cpxRA operon, encoding the sensor kinase and its cognate regulator CpxR, has no effect on division site biogenesis. It appears that certain mutant sensor kinases (CpxA*) either exhibit hyperactivity on CpxR or extend their signalling activity to one or more noncognate response regulators involved in cell division.     

Rhodopsin arginine-135 mutants are phosphorylated by rhodopsin kinase and bind arrestin in the absence of 11-cis-retinal

Arginine-135, located at the border between the third transmembrane domain and the second cytoplasmic loop of rhodopsin, is one of the most highly conserved amino acids in the family of G protein-coupled receptors. The effect of mutation at Arg-135 on the ability of rhodopsin to undergo desensitization was investigated. Four mutants, R135K, R135Q, R135A, and R135L, were examined for their ability to be phosphorylated by rhodopsin kinase, to bind arrestin, and to activate the rod cell G protein, transducin (Gt). All of the mutants were phosphorylated, bound arrestin, and were able to activate Gt when reconstituted with 11-cis-retinal. Surprisingly, several of the mutants could be phosphorylated by rhodopsin kinase and could bind arrestin in the absence of 11-cis-retinal but were not able to activate Gt. These observations represent the first demonstration of a mutant G protein-coupled receptor that assumes a conformation able to interact with its G protein-coupled receptor kinase and arrestin, but not with its G protein, in the absence of ligand.     

Parallel response selection disrupts sequence learning under dual-task conditions.

Some studies suggest that dual-task processing impairs sequence learning; others suggest it does not. The reason for this discrepancy remains obscure. It may have to do with the dual-task procedure often used. Many dual-task sequence learning studies pair the serial reaction time (SRT) task with a tone-counting secondary task. The tone-counting task, however, is not ideal for studying the cognitive processes involved in sequence learning. The present experiments sought to identify the nature of the interference responsible for disrupting sequence learning in dual-task situations using more tractable dual-task procedures. Experiments 1 and 2 showed that parallel-interfering central processing disrupts sequence learning. Experiment 3 used a novel combination of the SRT task as the secondary task in a psychological refractory period procedure. It showed that SRT task performance can be disrupted without disrupting sequence learning when that disruption involves a response-selection bottleneck rather than parallel response selection. Together, these results suggest that it is the overlap of central processes involved in successfully performing the 2 tasks concurrently that leads to learning deficits in dual-task sequence learning. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Predictive model selection for repeated measures random effects models using Bayes factors

The random effects model fit to repeated measures data is an extremely common model and data structure in current biostatistical practice. Modern data analysis often involves the selection of models within broad classes of prespecified models, but for models beyond the generalized linear model, few model-selection tools have been actively studied. In a Bayesian analysis, Bayes factors are the natural tool to use to explore these classes of models. In this paper, we develop a predictive approach for specifying the priors of a repeated measures random effects model with emphasis on selecting the fixed effects. The advantage of the predictive approach is that a single predictive specification is used to specify priors for all models considered. The methodology is applied to a pediatric pain data analysis.     

Alterations in topoisomerase IV and DNA gyrase in quinolone-resistant mutants of Mycoplasma hominis obtained in vitro

Mycoplasma hominis mutants were selected stepwise for resistance to ofloxacin and sparfloxacin, and their gyrA, gyrB, parC, and parE quinolone resistance-determining regions were characterized. For ofloxacin, four rounds of selection yielded six first-, six second-, five third-, and two fourth-step mutants. The first-step mutants harbored a single Asp426-->Asn substitution in ParE. GyrA changes (Ser83-->Leu or Trp) were found only from the third round of selection. With sparfloxacin, three rounds of selection generated 4 first-, 7 second-, and 10 third-step mutants. In contrast to ofloxacin resistance, GyrA mutations (Ser83-->Leu or Ser84-->Trp) were detected in the first-step mutants prior to ParC changes (Glu84-->Lys), which appeared only after the second round of selection. Further analysis of eight multistep-selected mutants of M. hominis that were previously described (2) revealed that they carried mutations in ParE (Asp426-->Asn), GyrA (Ser83-->Leu) and ParE (Asp426-->Asn), GyrA (Ser83-->Leu) and ParC (Ser80-->Ile), or ParC (Ser80-->Ile) alone, depending on the fluoroquinolone used for selection, i.e., ciprofloxacin, norfloxacin, ofloxacin, or pefloxacin, respectively. These data indicate that in M. hominis DNA gyrase is the primary target of sparfloxacin whereas topoisomerase IV is the primary target of pefloxacin, ofloxacin, and ciprofloxacin.     

Substrate-binding and catalytic roles of Lys194 in the C-terminus in human adenylate kinase by site-directed random mutagenesis

Site-directed random mutagenesis of Lys194 residue in the C-terminus of human adenylate kinase (AK) was performed, and six mutants were analyzed by steady-state kinetics. K194-mutants variously affected the apparent Michaelis constants (K(m) values) for ATP and AMP, although the kcat values strikingly decreased. The Lys194 residue appears to interact not only with MgATP2- but also with the AMP2- substrates by salt bridge formation with a nucleotide and to play a functional role in stabilizing the phosphate-transfer during catalysis. Lys194 could be essential for substrate-holdings and in catalysis and not replaceable to the other amino acids.     

Small circles of helical DNA obtained on the basis of transcriptional pause sites sequence

The 21-base pair synthetic DNA duplexes with basic 'pause-motif site ('CATGC') were ligated head-to-tail to produce linear and circular multimers. This also was done from other closely related sequences. Electrophoretic mobilities of the linear multimers in polyacrylamide gels were determined under the standard and modified conditions. We revealed that small linear multimers (approximately 90 bp) were characterized by comparable value of gel retardation relative to the well known curved DNA, while longer multimers (130 to approximately 170 bp) had only slightly expressed mobility anomaly. Nevertheless these multimers containing nontruncated 'pause-motif were capable of cyclization, in particular, formation of unusually small circles while truncated ones were not. We conclude that basic 'pause-motif site increases the closure ability while the multimers based on truncated 'pause motif fail to curve into the small circles. We tend to explain this situation as a result of intrinsic bending as well as the influence of the thermal fluctuations of DNA, the latter most probably can be associated with 'pause motif'. We have estimated the equilibrial and maximal bend angles per 10.5 bp to be 12 degrees to approximately 16 degrees and 32 degrees accordingly under experimental conditions of our study.     

Characterization of mutants affecting the KRK sequence in the carboxyl-terminal domain of lac repressor

The lac repressor carboxyl-terminal region is required for tetramer assembly and protein stability. To further investigate this region, especially the unusual sequence KRK, four deletion mutants eliminating the carboxyl-terminal 34, 35, 36, and 39 amino acids and five substitution mutants at the position of Arg-326, R326K, R326A, R326E, R326L, and R326W, were constructed using site-specific mutagenesis. The -34-amino-acid (aa) mutant, missing the most carboxyl-proximal lysine from the KRK sequence, exhibited lower affinity for both operator and inducer and lower protein stability than dimeric proteins studied previously. The -35-aa mutant with RK missing, as well as -36 aa and -39 aa, for which the entire KRK sequence was deleted, yielded inactive polypeptides that could be detected only by monoclonal antibody for lac repressor. In the Arg-326 mutant proteins, operator binding affinity was decreased by approximately 6-fold, the shift in inducer binding at elevated pH was diminished, and protein stability was decreased. Dramatic decreases in protein expression and stability occurred with substitution at position 326 by glutamate, leucine, or tryptophan. These results suggest that Arg-326 plays an important role in the formation of the proper tertiary structure necessary for inducer and operator affinity and for protein stability.     

Induction of CD4 expression and human immunodeficiency virus type 1 replication by mutants of the interferon-inducible protein kinase PKR

Replication of the human immunodeficiency virus type 1 (HIV-1) is inhibited by interferons (IFNs), and the IFN-inducible protein kinase PKR is thought to mediate this effect by regulating protein synthesis. Here we report that ectopic expression of dominant negative PKR mutants in Jurkat cells induces HIV-1 replication. Specifically, expression of CD4 is upregulated by the PKR mutants, and this correlates with an induction of HIV-1 binding and proviral DNA synthesis upon HIV-1 infection. Moreover, activation of NF-kappaB was induced by an RNA binding-defective mutant of PKR. Thus, it appears that PKR, in addition to translational control, is involved in HIV-1 replication by modulating virus binding through the regulation of CD4 expression and virus gene expression through the activation of NF-kappaB.     

DNA fingerprints of rice (Oryza sativa) obtained from hypervariable chloroplast simple sequence repeats

The aim of this research was to develop a convenient polymerase chain reaction-based assay that would allow intraspecific chloroplast variability to be detected. Our approach is based on the detection of length polymorphism within chloroplast mononucleotide microsatellite loci. Information from the fully sequenced rice chloroplast genome was used to identify 12 regions with a minimum of ten uninterrupted mononucleotide repeats. Primers flanking these repeats were used in conjunction with polymerase chain reaction to examine levels of polymorphism in six wild and 14 cultivated rice accessions. A total of six of the primer pairs revealed length polymorphism with between two and five size variants being detected. Diversity indices varied between 0.07 and 0.72. The length variation detected at multiple, physically linked sites was used to identify 15 unique haplotypes with an overall diversity index of 0.90. This level of polymorphism is sufficiently high to allow chloroplast variability to be studied at the intraspecific level. An additional 47 Oryza sativa accessions were also assayed with 31 unique chloroplast haplotypes being detected. The distribution of these haplotypes is described in relation to isozyme groupings and subspecies differentiation. The relevance and implications of these results for plant population genetics and the management of germplasm collections is discussed.     

In vivo selection of Rous sarcoma virus mutants with randomized sequences in the packaging signal

Retrovirus genomes contain a sequence at the 5' end which directs their packaging into virions. In Rous sarcoma virus, previous studies have identified important segments of the packaging signal, Psi, and support elements of a secondary-structure prediction. To further characterize this sequence, we used an in vivo selection strategy to test large collections of mutants. We generated pools of full-length viral DNA molecules with short stretches of random sequence in Psi and transfected each pool into avian cells. Resulting infectious virus was allowed to spread by multiple passages, so that sequences could compete and the best could be selected. This method provides information on the kinds of sequences allowed, as well as those that are most fit. Several predicted stem-loop structures in Psi were tested. A stem at the base of element O3 was highly favored; only sequences which maintained base pairing were selected. Two other stems, at the base and in the middle of element L3, were not conserved: neither base pairing nor sequence was maintained. A single mutation, G213U, was seen upstream of the randomized region in all selected L3 stem mutants; we interpret this to mean that it compensates for the defects in L3. Randomized mutations adjacent to G213 maintained the wild-type base composition but not its sequence. The kissing-loop sequence at end of L3, postulated to function in genome dimerization, was not required for infectivity but was selected for over time. Finally, a deletion of L3 was constructed and found to be poorly infectious.