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Results for “Selection, Genetic”

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PubMed2005

In vivo genetic selection of renal proximal tubules.

Repopulation by transplanted cells can result in effective therapy for several regenerative organs including blood, liver, and skin. In contrast, cell therapies for renal diseases are not currently available. Here we developed an animal model in which cells genetically resistant to a toxic intermediate of tyrosine metabolism, homogentisic acid (HGA), were able to repopulate the damaged proximal tubule epithelium of mice with fumarylacetoacetate hydrolase (Fah) deficiency. HGA resistance was achieved by two independent mechanisms. First, Fah+ transplanted bone marrow cells produced significant replacement of damaged proximal tubular epithelium (up to 50%). The majority of bone marrow-derived epithelial cells were generated by cell fusion, not transdifferentiation. In addition to regeneration by fusion-derived epithelial cells, proximal tubular repopulation was also observed by host epithelial cells, which had lost the homogentisic acid dioxygenase gene. These data demonstrate that extensive regeneration of the renal proximal tubule compartment can be achieved through genetic selection of functional cells.

Acute Disease↗

PubMedDate not supplied

Effects of T mitogens on in vivo antibody production to a T-dependent antigen in lines of mice genetically selected for high or low in vitro responsiveness to PHA.

The influence of genes which regulate the in vitro T-cell proliferative response to T mitogen upon in vivo antibody production to a T-dependent antigen was studied in two lines of mice genetically selected for a high or a low in vitro lymphocyte response to phytohaemagglutinin (PHA). Kinetics of agglutinin production to increasing doses of sheep erythrocytes was similar in the two lines, except for the titres of mercaptoethanol-resistant antibodies, which were slightly lower in the low-responder line. Treatment with mitogen prior to immunization modified the antibody response in the two lines differently. This finding would indicate that the genes which regulate in vitro stimulation of T cells by PHA also control in vivo activation of T-cell subsets involved in immunoresponsiveness.

Animals↗

PubMed1998

Differential sensory-motor effects of pentobarbital in intact rats genetically selected for high vs. low neuropathic pain-related behaviour.

Denervation of the hindpaw in rodents triggers autotomy, a behaviour of licking, scratching and self-mutilation of the denervated paw. This behaviour has been used as a model of paraesthesia, dysaesthesia and neuropathic pain. HA and LA rats are lines that have been genetically selected for high or low levels of autotomy, respectively. Compared to intact LA rats, HA rats are more sensitive to convulsions induced by pentylenetetrazol (PTZ), a blocker of the chloride channel associated with the GABA(A) receptor. Here we tested whether an acute administration of a sedative but not anaesthetic dose of pentobarbital (PB) would differentiate between these rat lines, in a number of sensory and motor tests performed in intact rats. This drug was tested since in contrast to PTZ, PB enhances central nervous system (CNS) inhibition by increasing chloride flux through the same channel. We found that PB was significantly more ataxic, antinociceptive, and reduced touch sensitivity in LA rats, compared to HA rats. These results suggest that HA and LA rats genetically differ in the levels of central inhibitions mediated by the GABA system presumably at the chloride channel. This difference may be associated with the dichotomous expression of neuropathic pain in these rat lines.

Animals↗

PubMed2002

Optimization of trans-splicing ribozyme efficiency and specificity by in vivo genetic selection.

Trans-splicing ribozymes are RNA-based catalysts capable of splicing RNA sequences from one transcript specifically into a separate target transcript. In doing so, a chimeric mRNA can be produced, and new gene activities triggered in living cells dependent on the presence of the target mRNA. Based on this ability of trans-splicing ribozymes to deliver new gene activities, a simple and versatile plating assay was developed in Saccharomyces cerevisiae for assessing and optimizing constructs in vivo. Trans-splicing ribozymes were used to splice sequences encoding a GAL4-derived transcription activator into a target transcript from a prevalent viral pathogen. The transcription activator translated from this new mRNA in turn triggered the expression of genes under the regulatory control of GAL4 upstream-activating sequences. Two of the activated genes complemented metabolic deficiencies in the host strain, and allowed growth on selective media. A simple genetic assay based on phenotypic conversion from auxotrophy to prototrophy was established to select efficient and specific trans-splicing ribozymes from a ribozyme library. This simple assay may prove valuable for selecting optimal target sites for therapeutic agents such as ribozymes, antisense RNA and antisense oligodeoxyribonucleotides, and for optimizing the design of the therapeutic agents themselves, in higher eukaryotes.

Cell Division↗

PubMed2004

Renin activity and angiotensin I concentration in genetically selective inbred line of hypertensive mice.

Blood pressure lowering kallikrein-kinin and blood pressure raising renin-angiotensin systems play a major role in the maintenance of normal blood pressure. In a previous study, we have shown that a kallikrein-like prorenin converting enzyme (PRCE C) is elevated in the submandibular gland tissue of a mouse line (BPH) that was genetically selected and inbred for high blood pressure in comparison to normotensive line (BPN) that was derived from the ancestors of BPH line. In the present investigation we wanted to find out if elevated levels of PRCE C were involved in the modulation of tissue (local) renin-angiotensin system in the submandibular gland tissue. Results indicate significantly high renin activity but low angiotensin I level in the tissue of BPH mouse model. These results tend to suggest PRCE C's involvement in tissue (local) renin-angiotensin system.

Angiotensin I↗

PubMed1991

Direct genetic selection of a maize cDNA for dihydrodipicolinate synthase in an Escherichia coli dapA- auxotroph.

Dihydrodipicolinate synthase (DHPS; EC 4.2.1.52) is the first committed enzyme in the lysine branch of the aspartate-derived amino acid biosynthesis pathway and is common to bacteria and plants. Due to feedback inhibition by lysine, DHPS serves in a regulatory role for this pathway in plant metabolism. To elucidate the molecular genetic characteristics of DHPS, we isolated a putative full-length cDNA clone for maize DHPS by direct genetic selection in an Escherichia coli dapA- auxotroph. The maize DHPS activity expressed in the complemented E. coli auxotroph showed the lysine inhibition characteristics of purified maize DHPS, indicating that the cDNA encoded sequences for both the catalytic function and regulatory properties of the enzyme. The N-terminal amino acid sequence of purified maize DHPS was determined by direct sequencing and showed homology to a sequence within the cDNA, indicating that the clone contained the entire coding region for a mature polypeptide of 326 amino acids plus a 54 amino acid transit peptide sequence. The molecular weight of 35,854, predicted from the deduced amino acid sequence, was similar to the 38,000 Mr determined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) for the purified enzyme from maize. DHPS mRNAs complementary to the cDNA were detected in RNA isolated from developing maize endosperm and embryo tissues. Southern blots indicated the presence of more than one genomic sequence homologous to DHPS per haploid maize genome.

Amino Acid Sequence↗

PubMed2005

Effect of genetic selection on MyoD and myogenin expression in turkeys with different growth rates.

Skeletal muscle development requires the ordered expression of specific myogenic regulatory factors, which include MyoD, Myf5, myogenin, and MRF4. The MyoD and Mrf5 factors are required for the determination of myoblasts, whereas myogenin and MRF4 play a pivotal role in terminal differentiation. In the current study, males and females of a turkey genetic line selected only for increased 16-wk BW (F line) and an unselected randombred control (RBC2 line) from which the F line was developed were used to investigate the developmental expression of MyoD and myogenin mRNA in embryonic pectoralis major muscle and myogenic satellite cells. Pectoralis major muscle was isolated at embryonic d (ED) 14, 16, 18, 20, 22, and 24. The mRNA levels of MyoD and myogenin were measured using a real-time quantitative polymerase chain reaction method. Both MyoD and myogenin expression declined during embryonic development. The decrease in MyoD expression started at ED 16 for the F line and at ED 18 for the RBC2 line for both sexes. Myogenin expression in both lines began to decline at ED 14. The F line males had lower myogenin expression at ED 14, 16, and 18 than the RBC2 line males, which was similar for the F line females compared with the RBC2 line females except there was no significant difference at ED 18. The RBC2 line males had greater expression than the females for myogenin at ED 16 and 18 for the RBC2 line. Proliferating myogenic satellite cells in both lines and sexes expressed low levels of MyoD and myogenin. After the initiation of differentiation in both lines and sexes, there was a sharp surge in MyoD expression at 24 h followed by a decrease at 48 h and then an increase in expression through 72 or 96 h of differentiation. There were line and sex differences in myogenin expression during the differentiation process. These data are suggestive of growth- and sex-related differences in the expression of myogenic regulatory factors key to muscle cell proliferation and differentiation, which will affect muscle growth rate.

Animals↗

PubMed1995

Increased expression and activity of ornithine decarboxylase in chicks genetically selected for rapid growth rate.

Polyamines are essential requirements for cell proliferation and their role in stimulation of RNA, DNA, and protein synthesis is clearly established. Ornithine decarboxylase is a key enzyme in the biosynthesis of the polyamines and its activity is regulated in response to factors that stimulate cell proliferation. Steady-state ODC mRNA levels and enzyme activity were measured in muscle of chicks genetically selected for increased growth rate or for egg production. In muscle, muscle satellite cells and myotubes, two ODC mRNA transcripts are present of molecular size 2.05 and 1.75 kb. Northern blotting analysis suggest that these transcripts are produced as a result of using different polyadenylation sites. Between day 1 and day 6 after hatching, a period of rapid muscle growth in these animals, a peak in muscle ODC mRNA levels is followed by a peak in enzyme activity in both lines. Significantly higher ODC mRNA levels and enzyme activity are associated with selection for rapid growth in the broiler line. The results are consistent with other data showing that ODC is a major factor in cell growth and provide further evidence that it is a candidate 'trait-gene' for growth.

Animals↗

PubMed2005

An intein-based genetic selection allows the construction of a high-quality library of binary patterned de novo protein sequences.

Combinatorial libraries of synthetic DNA are increasingly being used to identify and evolve proteins with novel folds and functions. An effective strategy for maximizing the diversity of these libraries relies on the assembly of large genes from smaller fragments of synthetic DNA. To optimize library assembly and screening, it is desirable to remove from the synthetic libraries any sequences that contain unintended frameshifts or stop codons. Although genetic selection systems can be used to accomplish this task, the tendency of individual segments to yield misfolded or aggregated products can decrease the effectiveness of these selections. Furthermore, individual protein domains may misfold when removed from their native context. We report the development and characterization of an in vivo system to preselect sequences that encode uninterrupted gene segments regardless of the foldedness of the encoded polypeptide. In this system, the inserted synthetic gene segment is separated from an intein/thymidylate synthase (TS) reporter domain by a polyasparagine linker, thereby permitting the TS reporter to fold and function independently of the folding and function of the segment-encoded polypeptide. TS-deficient Escherichia coli host cells survive on selective medium only if the insert is uninterrupted and in-frame, thereby allowing selection and amplification of desired sequences. We demonstrate that this system can be used as a highly effective preselection tool for the production of large, diverse and high-quality libraries of de novo protein sequences.

Amino Acid Sequence↗

PubMed2004

Degenerate in vitro genetic selection reveals mutations that diminish alfalfa mosaic virus RNA replication without affecting coat protein binding.

The alfalfa mosaic virus (AMV) RNAs are infectious only in the presence of the viral coat protein; however, the mechanisms describing coat protein's role during replication are disputed. We reasoned that mechanistic details might be revealed by identifying RNA mutations in the 3'-terminal coat protein binding domain that increased or decreased RNA replication without affecting coat protein binding. Degenerate (doped) in vitro genetic selection, based on a pool of randomized 39-mers, was used to select 30 variant RNAs that bound coat protein with high affinity. AUGC sequences that are conserved among AMV and ilarvirus RNAs were among the invariant nucleotides in the selected RNAs. Five representative clones were analyzed in functional assays, revealing diminished viral RNA expression resulting from apparent defects in replication and/or translation. These data identify a set of mutations, including G-U wobble pairs and nucleotide mismatches in the 5' hairpin, which affect viral RNA functions without significant impact on coat protein binding. Because the mutations associated with diminished function were scattered over the 3'-terminal nucleotides, we considered the possibility that RNA conformational changes rather than disruption of a precise motif might limit activity. Native polyacrylamide gel electrophoresis experiments showed that the 3' RNA conformation was indeed altered by nucleotide substitutions. One interpretation of the data is that coat protein binding to the AUGC sequences determines the orientation of the 3' hairpins relative to one another, while local structural features within these hairpins are also critical determinants of functional activity.

3' Untranslated Regions↗

PubMed1984

Relationship of brown adipose tissue with growth and obesity differences in genetically selected mouse lines.

A recent hypothesis considers brown adipose tissue (BAT) to be an important source of diet-induced thermogenesis (DIT). In turn, DIT and thermogenesis in general are believed to be key factors in the control of obesity of laboratory rodents. This hypothesis was developed from the study of single gene mutant obese rodents. The present research tested this hypothesis in mice with polygenic control of growth and obesity, which is more characteristic of the type of genetic variation expected in human and other mammalian populations. Control and high fat diets were used to test responses of five genetically selected lines of mice showing different patterns of growth and obesity. All lines deposited more fat on the high fat diet, but the most obese line showed the largest increase in BAT and the lipid-free dry (LFD) component of BAT. Use of LFD per unit body weight gave results which supported the hypothesis being tested, but it was argued that this measure is misleading. When brown and white adipose tissue growth relative to body weight were examined, 2 of the 10 line-diet groups showed alterations in BAT growth patterns. However, it was concluded that BAT, if involved at all, was not a major factor in growth and obesity differences.

Adipose Tissue, Brown↗

PubMed2006

Genetic selection for protein solubility enabled by the folding quality control feature of the twin-arginine translocation pathway.

One of the most vexing problems facing structural genomics efforts and the biotechnology enterprise in general is the inability to efficiently produce functional proteins due to poor folding and insolubility. Additionally, protein misfolding and aggregation has been linked to a number of human diseases, such as Alzheimer's. Thus, a robust cellular assay that allows for direct monitoring, manipulation, and improvement of protein folding could have a profound impact. We report the development and characterization of a genetic selection for protein folding and solubility in living bacterial cells. The basis for this assay is the observation that protein transport through the bacterial twin-arginine translocation (Tat) pathway depends on correct folding of the protein prior to transport. In this system, a test protein is expressed as a tripartite fusion between an N-terminal Tat signal peptide and a C-terminal TEM1 beta-lactamase reporter protein. We demonstrate that survival of Escherichia coli cells on selective medium expressing a Tat-targeted test protein/beta-lactamase fusion correlates with the solubility of the test protein. Using this assay, we isolated solubility-enhanced variants of the Alzheimer's Abeta42 peptide from a large combinatorial library of Abeta42 sequences, thereby confirming that our assay is a highly effective selection tool for soluble proteins. By allowing the bacterial Tat pathway to exert folding quality control on expressed target protein sequences, we have generated a powerful tool for monitoring protein folding and solubility in living cells, for molecular engineering of solubility-enhanced proteins or for the isolation of factors and/or cellular conditions that stabilize aggregation-prone proteins.

Amino Acid Sequence↗

PubMed1999

Genetic selection of mutations in the high affinity K+ transporter HKT1 that define functions of a loop site for reduced Na+ permeability and increased Na+ tolerance.

Potassium is an important macronutrient required for plant growth, whereas sodium (Na+) can be toxic at high concentrations. The wheat K+ uptake transporter HKT1 has been shown to function in yeast and oocytes as a high affinity K+-Na+ cotransporter, and as a low affinity Na+ transporter at high external Na+. A previous study showed that point mutations in HKT1, which confer enhancement of Na+ tolerance to yeast, can be isolated by genetic selection. Here we report on the isolation of mutations in new domains of HKT1 showing further large increases in Na+ tolerance. By selection in a Na+ ATPase deletion mutant of yeast that shows a high Na+ sensitivity, new HKT1 mutants at positions Gln-270 and Asn-365 were isolated. Several independent mutations were isolated at the Asn-365 site. N365S dramatically increased Na+ tolerance in yeast compared with all other HKT1 mutants. Cation uptake experiments in yeast and biophysical characterization in Xenopus oocytes showed that the mechanisms underlying the Na+ tolerance conferred by the N365S mutant were: reduced inhibition of high affinity Rb+ (K+) uptake at high Na+ concentrations, reduced low affinity Na+ uptake, and reduced Na+ to K+ content ratios in yeast. In addition, the N365S mutant could be clearly distinguished from less Na+-tolerant HKT1 mutants by a markedly decreased relative permeability for Na+ at high Na+ concentrations. The new mutations contribute to the identification of new functional domains and an amino acid in a loop domain that is involved in cation specificity of a plant high affinity K+ transporter and will be valuable for molecular analyses of Na+ transport mechanisms and stress in plants.

Adaptation, Physiological↗

PubMed1999

Genetic selection of short peptides that support protein oligomerization in vivo.

An important goal in protein engineering is to control associations between designed proteins. This is most often done by fusing known, naturally occurring oligomerization modules, such as leucine zippers [1] [2] [3], to the proteins of interest [4] [5] [6]. It is of considerable interest to design or discover new oligomerization domains that have novel binding specificities [7] [8] [9] [10] [11] in order to expand the 'toolbox' of the protein engineer and also to eliminate associations of the designed proteins with endogenous factors. We report here a simple genetic selection scheme through which to search libraries for peptides that are able to mediate homodimerization or higher-order self-oligomerization of a protein in vivo. We found several peptides that support oligomerization of the lambda repressor DNA-binding domain in Escherichia coli cells, some of them as efficiently as the endogenous dimerization domain or the GCN4 leucine zipper. Many are very small, comprising as few as six residues. This study strongly supports the notion that peptide sequence space is rich in small peptides, which might be useful in protein engineering and other applications.

Amino Acid Sequence↗

PubMed1997

In vitro genetic selection analysis of alfalfa mosaic virus coat protein binding to 3'-terminal AUGC repeats in the viral RNAs.

The coat proteins of alfalfa mosaic virus (AMV) and the related ilarviruses bind specifically to the 3' untranslated regions of the viral RNAs, which contain conserved repeats of the tetranucleotide sequence AUGC. The purpose of this study was to develop a more detailed understanding of RNA sequence and/or structural determinants required for coat protein binding by characterizing the role of the AUGC repeats. Starting with a complex pool of 39-nucleotide RNA molecules containing random substitutions in the AUGC repeats, in vitro genetic selection was used to identify RNAs that bound coat protein. After six iterative rounds of selection, amplification, and reselection, 25% of the RNAs selected from the randomized pool were wild type; that is, they contained all four AUGC sequences. Among the 31 clones analyzed, AUGC was clearly the preferred selected sequence at the four repeats, but some nucleotide sequence variability was observed at AUGC(865-868) if the other three AUGC repeats were present. Variant RNAs that bound coat protein with affinities equal to or greater than that of the wild-type molecule were not selected. To extend the in vitro selection results, RNAs containing specific nucleotide substitutions were transcribed in vitro and tested in coat protein and peptide binding assays. The data strongly suggest that the AUGC repeats provide sequence-specific determinants and contribute to a structural platform for specific coat protein binding. Coat protein may function in maintaining the 3' ends of the genomic RNAs during replication by stabilizing an RNA structure that defines the 3' terminus as the initiation site for minus-strand synthesis.

Alfalfa mosaic virus↗

PubMed2004

A genetic selection for circadian output pathway mutations in Neurospora crassa.

In most organisms, circadian oscillators regulate the daily rhythmic expression of clock-controlled genes (ccgs). However, little is known about the pathways between the circadian oscillator(s) and the ccgs. In Neurospora crassa, the frq, wc-1, and wc-2 genes encode components of the frq-oscillator. A functional frq-oscillator is required for rhythmic expression of the morning-specific ccg-1 and ccg-2 genes. In frq-null or wc-1 mutant strains, ccg-1 mRNA levels fluctuate near peak levels over the course of the day, whereas ccg-2 mRNA remains at trough levels. The simplest model that fits the above observations is that the frq-oscillator regulates a repressor of ccg-1 and an activator of ccg-2. We utilized a genetic selection for mutations that affect the regulation of ccg-1 and ccg-2 by the frq-oscillator. We find that there is at least one mutant strain, COP1-1 (circadian output pathway derived from ccg-1), that has altered expression of ccg-1 mRNA, but normal ccg-2 expression levels. However, the clock does not appear to simply regulate a repressor of ccg-1 and an activator of ccg-2 in two independent pathways, since in our selection we identified three mutant strains, COP1-2, COP1-3, and COP1-4, in which a single mutation in each strain affects the expression levels and rhythmicity of both ccg-1 and ccg-2.

Blotting, Northern↗

PubMed2001

Enhanced antitumor efficacy of a herpes simplex virus mutant isolated by genetic selection in cancer cells.

Replication-competent, attenuated herpes simplex virus-1 (HSV-1) derivatives that contain engineered mutations into the viral gamma 34.5 virulence gene have been used as oncolytic agents. However, as attenuated mutants often grow poorly, they may not completely destroy some tumors and surviving cancer cells simply regrow. Thus, although HSV-1 gamma 34.5 mutants can reduce the growth of human tumor xenografts in mice and have passed phase I safety studies, their efficacy is limited because they replicate poorly in many human tumor cells. Previously, we selected for a gamma 34.5 deletion mutant variant that regained the ability to replicate efficiently in tumor cells. Although this virus contains an extragenic suppressor mutation that confers enhanced growth in tumor cells, it remains attenuated. Here, we demonstrate that the suppressor virus replicates to greater levels in prostate carcinoma cells and, importantly, is a more potent inhibitor of tumor growth in an animal model of human prostate cancer than the gamma 34.5 parent virus. Thus, genetic selection in cancer cells can be used as a tool to enhance the antitumor activity of a replication-competent virus. The increased therapeutic potency of this oncolytic virus may be useful in the treatment of a wide variety of cancers.

Adenocarcinoma↗

PubMed2006

Genetic selection for resistance or susceptibility to oral tolerance imparts correlation to both Immunoglobulin E level and mast cell number phenotypes with a profound impact on the atopic potential of the individual.

BACKGROUND: Immunological oral tolerance is being studied with great interest due to its therapeutic potential in allergy and autoimmunity processes, although the cellular and molecular mechanisms linking these different phenomena remain elusive. In the present study, two mouse lines with extreme phenotypes for susceptibility [TS Line] or resistance [TR Line] to oral tolerance and their [TS x TR]F2 segregants were used in order to evaluate the impact of these traits on the atopic potential of the individuals. OBJECTIVE: Demonstrate whether the tr and ts genes, cumulated during 18 generations of bidirectional genetic selection, influence expression of two important immunobiological traits (IgE and mast cell) critical to allergic response. METHODS: Mice with extreme phenotypes for oral tolerance to ovalbumin (OVA), produced by assortative mating (TS and TR Line), and their (TS x TR)F2 segregating were used. Serum IgE levels assayed by ELISA, and mastocytes counted with toluidine blue staining were evaluated in naïve mice. Anaphylaxis was induced by intravenous injection of OVA, intestinal inflammation by oral administration of OVA 7 days after immunization, and pulmonary inflammation by intranasal and nebulization OVA challenges. Specific IgE was dosed by passive cutaneous anaphylaxis. RESULTS: The naïve TS mice have a 20-fold lower serum IgE level and two- to threefold diminished mast cell numbers in mucosal sites, when compared with TR-mice, which were highly susceptible to allergic inflammation and anaphylactic shock. The associations of oral tolerance, serum IgE levels and mast cell numbers in naïve animals were confirmed analysing the simultaneous presence of these traits in individuals of a [TS x TR]F2 -segregating population. CONCLUSION: The results suggest that the complex of genes controlling TS and TR phenotypes play a main role in the regulation of the atopic potential of the individual. The studies of these traits in interline F2 segregants demonstrated a co-segregation of TS and TR phenotypes with IgE responsiveness and mast cell numbers. Thus, the opposite capacity of the genetically modified mice may be involved in co-adaptative mechanisms reflecting a dynamic relation between gene frequencies in a natural population. These correlations give circumstantial evidence to support clinical applications of oral tolerance in allergic and autoimmune diseases.

Administration, Oral↗