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Results for “Selection, Genetic”

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PubMed1997

Estrogen receptor is not primarily responsible for altered responsiveness of ovalbumin mRNA induction in the oviduct from genetically selected high- and low-albumen chicken lines.

The role of estrogen receptor on ovalbumin mRNA induction by steroid hormones was investigated in primary cultures of oviduct cells from estrogen-stimulated immature chicks of genetically selected high- and low-albumen egg laying lines (H- and L-lines). In experiment 1, the extent of ovalbumin mRNA induction and changes in estrogen and progesterone receptors were compared between the oviduct cells from H- and L-lines with or without steroid hormones in the culture medium. In experiment 2, the effect of estrogen receptor gene transfection on the induction of ovalbumin mRNA was studied in the oviduct cells from the L-line chicks. The results showed a close correlation of the changes in ovalbumin mRNA with the numbers of nuclear and total estrogen receptors in the oviduct cells but not with the numbers of nuclear and total progesterone receptors. Estrogen receptor gene transfection induced ovalbumin mRNA to a moderate extent in the absence of the steroid hormones. To our surprise, however, estrogen receptor gene transfection apparently suppressed the ovalbumin mRNA responsiveness to estrogen to a considerable extent. It was concluded, therefore, that the extent of estrogen receptor expression might not be primarily responsible for the differences in responsiveness to steroid hormones of oviduct cells from genetically selected H- and L-line chickens.

Animals↗

PubMed1991

The effects of genetic selection for increased growth rate on mucosal and muscle weights in the different regions of the small intestine of the domestic fowl (Gallus domesticus).

1. The total organ weights and the constituent weights of mucosal and muscle tissues have been determined in the small intestines of three lines of domestic fowls subjected to different degrees of artificial genetic selection for rapid growth rates. 2. Effects of selection have been examined on the basis of wet and dry tissue weights in duodenum, jejunum and ileum from each line of birds. 3. Effects of selection have been compared on both absolute tissue weights and relative weights (i.e. expressed per kilogram body weight). 4. Selection for rapid growth rate is associated with increases in absolute weight and length of small intestine but marked reductions in relative weight and length. 5. In the fastest growing group of birds the major effect is a decrease in the relative mass of mucosa which is most pronounced in the more proximal regions of the small intestine. 6. These findings are discussed in relation to possible enhancement of intestinal digestive and absorptive efficiency in birds selected for improved growth rate and feed conversion. 7. The differences in tissue composition of the small intestine between lines and between regions of the intestine in the same line emphasise the difficulty in selecting an anatomical parameter upon which to base the in vitro and in vivo measurements of nutrient absorption in a number of established preparations.

Animals↗

PubMed1999

Analysis of estrogen response element binding by genetically selected steroid receptor DNA binding domain mutants exhibiting altered specificity and enhanced affinity.

To analyze the role of amino acids in the steroid receptor DNA binding domain (DBD) recognition helix in binding of the receptor to the estrogen response element (ERE), we adapted the powerful P22 challenge phage selection system for use with a vertebrate protein. We used the progesterone receptor DNA binding domain and selected for mutants that gained the ability to bind to the ERE. We used a mutagenesis protocol based on degenerate oligonucleotides to create a large and diverse pool of mutants in which 10 nonconsensus amino acids in the DNA recognition helix of the progesterone receptor DNA binding domain were randomly mutated. After a single cycle of modified P22 challenge phage selection, 37 mutant proteins were identified, all of which lost the ability to bind to the progesterone response element. In gel mobility shift assays, approximately 70% of the genetically selected mutants bound to the consensus ERE with a >4-fold higher affinity than the naturally occurring estrogen receptor DBD. In the P-box region of the DNA recognition helix, the selected mutants contained the amino acids found in the wild-type estrogen receptor DBD, as well as other amino acid combinations seen in naturally occurring steroid/nuclear receptors that bind the aGGTCA half-site. We also obtained high affinity DBDs with Trp(585) as the first amino acid of the P-box, although this is not found in the known steroid/nuclear receptors. In the linker region between the two zinc fingers, G597R was by far the most common mutation. In transient transfections in mammalian cells using promoter interference assays, the mutants displayed enhanced affinity for the ERE. When linked to an activation domain, the transfected mutants activated transcription from ERE-containing reporter genes. We conclude that the P-box amino acids can display considerable variation and that the little studied linker amino acids play an important role in determining affinity for the ERE. This work also demonstrates that the P22 challenge phage genetic selection system, modified for use with a mammalian protein, provides a novel, single cycle selection for steroid/nuclear receptor DBDs with altered specificity and greatly enhanced affinity for their response elements.

Amino Acid Sequence↗

PubMed1993

Recombination walking: genetic selection of clones from pooled libraries of yeast artificial chromosomes by homologous recombination.

Recombination walking is based on the genetic selection of specific human clones from a yeast artificial chromosome (YAC) library by homologous recombination. The desired clone is selected from a pooled (unordered) YAC library, eliminating labor-intensive steps typically used in organizing and maintaining ordered YAC libraries. Recombination walking represents an efficient approach to library screening and is well suited for chromosome-walking approaches to the isolation of genes associated with common diseases.

Animals↗

PubMed1984

Female sex hormone and nephropathy in Cohen diabetic rat (genetically selected sucrose-fed).

In the Cohen diabetic rat (genetically-selected sucrose-fed) ovariectomy resulted in a significantly decreased incidence of nephropathy, while estradiol treatment to the ovariectomized animal increased the rate of nephropathy to the levels occurring in the uncastrated untreated diabetic female. In the male diabetic animal orchidectomy caused a slight decrease in the rate of nephropathy whereas estradiol treatment to the orchidectomized animal resulted in a slight increase in the rate. No difference was found between the ovariectomized and the uncastrated diabetic female as to the spontaneous blood glucose, plasma insulin, serum cholesterol or TG levels, but a significant difference was found in their plasma estradiol levels.

Animals↗

PubMed1994

Mapping critical residues in eukaryotic DNA-binding proteins: a plasmid-based genetic selection strategy with application to the Oct-2 POU motif.

Discrimination between allowed and disallowed amino acid substitutions provides a powerful method for analysis of protein structure and function. Site-directed mutagenesis allows specific hypotheses to be tested, but its systematic application to entire structural motifs is inefficient. This limitation may be overcome by genetic selection, which allows rapid scoring of thousands of randomly (or pseudorandomly) generated mutants. To facilitate structural dissection of DNA-binding proteins, we have designed two generally applicable bacterial selection systems: pPLUS selects for the ability of a protein to bind to a user-defined DNA sequence, whereas pMINUS selects against such binding. Complementary positive and negative selections allow rapid mapping of critical residues. To test and calibrate the systems, we have investigated the bipartite POU domain of the human B-cell-specific transcription factor Oct-2. (i) An invariant residue (Asn347) in the DNA-recognition helix of the POU-specific homeodomain (POUHD) was substituted by each of the 19 other possible amino acids. The mutant proteins each exhibited decreased specific DNA binding as defined in vivo by genetic selection and in vitro by gel retardation; relative affinities correlate with phenotypes in the positive and negative selection systems. An essential role for Asn347 in wild-type POUHD-DNA recognition is consistent with homologous Asn-adenine interactions in cocrystal structures of canonical homeodomains. (ii) Extension of pPLUS/pMINUS selection to the POU-specific subdomain (POUs) is demonstrated by analysis of mutations in its putative helix-turn-helix (HTH) element.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗

PubMed2001

Rapid genetic selection of inhibitor-resistant protease mutants: clinically relevant and novel mutants of the HIV protease.

Site-specific proteolysis is an important regulatory mechanism in many basic cellular processes as well as playing critical roles in the life cycle of viruses and other pathogenic organisms. For the human immunodeficiency virus (HIV) the encoded protease is required for the replication of the virus and has been the target of novel antiviral therapeutics. However, the emergence of inhibitor-resistant viral strains has become an increasingly significant clinical problem. Using a bacteriophage-based genetic selection, a bank of inhibitor-resistant mutants in this protease has been isolated that includes mutations that correlate with resistant clinical isolates. The rapid selection of such mutations has implications for the prediction of relevant mutations and may be applicable to other viral systems.

Bacteriophage lambda↗

PubMed2002

Genetic selection of peptide inhibitors of human immunodeficiency virus type 1 Vpr.

Human immunodeficiency virus 1 (HIV-1) encodes a gene product, Vpr, that facilitates the nuclear uptake of the viral pre-integration complex in non-dividing cells and causes infected cells to arrest in the G(2) phase of the cell cycle. Vpr was also shown to cause mitochondrial dysfunction in human cells and budding yeasts, an effect that was proposed to lead to growth arrest and cell killing in budding yeasts and apoptosis in human cells. In this study, we used a genetic selection in Saccharomyces cerevisiae to identify hexameric peptides that suppress the growth arrest phenotype mediated by Vpr. Fifteen selected glutathione S-transferase (GST)-fused peptides were found to overcome to different extents Vpr-mediated growth arrest. Amino acid analysis of the inhibitory peptide sequences revealed the conservation of a di-tryptophan (diW) motif. DiW-containing GST-peptides interacted with Vpr in GST pull-down assays, and their level of interaction correlated with their ability to overcome Vpr-mediated growth arrest. Importantly, Vpr-binding GST-peptides were also found to alleviate Vpr-mediated apoptosis and G(2) arrest in HIV-1-producing CD4(+) T cell lines. Furthermore, they co-localized with Vpr and interfered with its nuclear translocation. Overall, this study defines a class of diW-containing peptides that inhibit HIV-1 Vpr biological activities most likely by interacting with Vpr and interfering with critical protein interactions.

Animals↗

PubMed1982

Some applications of inequalities for extreme order statistics to a genetic selection problem.

Two inequalities are derived for the extreme order statistics in a special case of unequally-correlated multivariate normal variables, and their implications in a genetic selection problem which was considered recently by Rawlings (1976, Biometrics 32, 875-887) and Hill (1976, Biometrics 32, 889-902; 1977, Biometrics 33, 703-712) are discussed. The inequalities establish a monotonicity property of the extreme order statistics as a function of the intraclass correlation, and they yield a chain of bounds when the family sizes are not all equal. Thus the inequalities extend the applicability of the results given by Rawlings and Hill to the more general case.

Animals↗

PubMed1994

Novel estrogen response elements identified by genetic selection in yeast are differentially responsive to estrogens and antiestrogens in mammalian cells.

A powerful and versatile system for the identification of novel response elements for members of the intracellular receptor family is presented as applied to the human estrogen receptor. In the past, a limited number of estrogen response elements (EREs) have been functionally identified in the promoter regions of estrogen-regulated genes. From these a consensus ERE has been defined that is identical to the ERE of the Xenopus laevis vitellogenin gene, i.e., 5'-GGTCA NNN TGACC-3'. In order to investigate without bias the range of sequences that could function as EREs in vivo, we have developed a genetic selection in yeast expressing the human estrogen receptor (hER) and transformed with a random oligonucleotide library in a vector where expression of a selectable marker requires insertion of an upstream activating sequence. More than 1,000,000 transformants were screened and of 726 clones that contained activating sequences, 65 were found to be hormone-dependent. Sequencing revealed that the majority contained at least one 4/5 match to a canonical ERE half-site, but only one contained a full consensus ERE as previously defined. Some contained half-sites arranged as direct repeats. Twelve elements were further characterized to compare estrogen activation in yeast and mammalian cells and in vitro binding to hER. The results of these studies reveal that sequences that bind weakly to hER in vitro are fully functional as EREs in yeast and are conditionally responsive to estrogen in mammalian cells. In addition, an element was identified that is more sensitive to the partial agonist activities of tamoxifen and nafoxidine than is the consensus ERE, indicating that not only promoter context but the sequence of the binding site itself can allow distinction between receptor activated by agonist and that activated by antagonist.

Base Sequence↗

PubMed1981

The Bethlem lines: genetic selection for high and low rearing activity in rats.

A foundation population derived from nine different rat strains was subjected to genetic selection for high and low rearing activity. A rapid response to selection was demonstrated, the two selected lines showing virtually no overlap in scores after 10 generations. Heritability estimates were assessed from parent-offspring regressions in the unselected control line and from comparisons of the achieved response with the selection pressure applied in the selected lines. Overall, the heritability of rearing activity in the rat was assessed to be about 0.25. In addition, a major gene effect was revealed, albinism being associated with low rearing activity scores.

Animals↗

PubMed1989

Positive genetic selection for gene disruption in mammalian cells by homologous recombination.

Efficient modification of genes in mammalian cells by homologous recombination has not been possible because of the high frequency of nonhomologous recombination. An efficient method for targeted gene disruption has been developed. Cells with substitution of exogenous sequences into a chromosomal locus were enriched, by a factor of 100, using a positive genetic selection that specifically selects for homologous recombination at the targeted site. The selection is based on the conditional expression of a dominant selectable marker by virtue of in-frame gene fusion with the target gene. The dominant selectable marker was derived by modification of the Escherichia coli neo gene so that it retains significant activity in mammalian cells after in-frame fusion with heterologous coding sequences. In the example presented here, homologous recombinants were efficiently recovered from a pool in which the targeted gene was disrupted in 1 per 10,000 cells incorporating exogenous DNA.

Animals↗

PubMed2000

Increased hsp22 RNA levels in Drosophila lines genetically selected for increased longevity.

RNAs for the small heat shock protein (hsp) genes hsp22 and hsp23 are induced during Drosophila aging, suggesting that these genes might have specific functions at late ages. To determine if hsp22 and hsp23 gene expression might correlate with life span, RNA levels for these and additional genes were analyzed throughout the adult life span in a set of five outbred "O" lines, which have been genetically selected for increased longevity, and in five matched control "B" lines. Control ribosomal protein genes rp49 and AP3/RpPO RNA levels were similar in O and B lines. In contrast, hsp22 RNA levels were twofold-tenfold higher in all five O lines relative to all five B lines, while hsp23 exhibited a smaller but significant increase. Thus increased hsp22 and hsp23 RNA levels correlate with the increased life span and increased stress resistance of the genetically selected O lines.

Animals↗

PubMed2005

Effects of week of lactation and genetic selection for milk yield on milk fatty acid composition in Holstein cows.

Control (CL) and select line (SL) dairy cows (n = 22) managed identically but differing in milk yield (>4100 kg/305 d) were used to determine differences in milk fatty acid profile as lactation progressed. Milk yield was recorded daily and milk samples were collected during wk 1, 4, 8, 12, and 16 postpartum for milk composition analysis. Milk samples from wk 1, 8, and 16 were also analyzed for fatty acid composition. Select-line cows produced more milk (44.4 vs. 31.2 kg/d) and milk components than CL cows during the 16-wk period. There was no difference in rate of milk yield increase, but peak milk yield for SL cows was greater and occurred later in lactation. There were no differences in milk SCC or milk fat, protein, or lactose content. Selection for milk yield did not affect the content of most individual milk fatty acids; however, compared with CL, SL cows had a reduced Delta(9)-desaturase system and tended to produce milk with lower monounsaturated fatty acid content. Selection for milk yield did not affect milk fatty acid origin but the percentage of de novo fatty acids increased and preformed fatty acids decreased as lactation progressed. Milk fat trans-11 18:1 and cis-9,trans-11 conjugated linoleic acid increased with progressing lactation (10.7 vs. 14.1 and 3.1 vs. 5.4 mg/g, or 31 and 76%, respectively) and were correlated strongly among wk 1, 8, and 16 of lactation. Temporal changes in the Delta(9)-desaturase system occurred during lactation but these changes were not correlated with milk fat cis-9,trans-11 conjugated linoleic acid content. Results indicate prolonged genetic selection for milk yield had little effect on milk fatty acid composition, but milk fatty acid profiles varied markedly by week of lactation.

Animals↗

PubMed2001

T7 phage display: a novel genetic selection system for cloning RNA-binding proteins from cDNA libraries.

RNA-binding proteins are central to posttranscriptional gene regulation and play an important role in a number of major human diseases. Cloning such proteins is a crucial but often difficult step in elucidating the biological function of RNA regulatory elements. To make it easier to clone proteins that specifically bind RNA elements of interest, we have developed a rapid and broadly applicable in vitro genetic selection method based on T7 phage display. Using hairpin II of U1 small nuclear RNA (U1hpII) or the 3' stem loop of histone mRNA as bait, we could selectively amplify T7 phage that display either the spliceosomal protein U1A or the histone stem loop-binding protein from a lung cDNA phage library containing more than 10(7) independent clones. The use of U1hpII mutants with various affinities for U1A revealed that this method allows the selection even of proteins that bind their cognate RNA targets with relatively weak affinities (K(d) as high as the micromolar range). Experiments with a mixture of recombinant phage displaying U1A or the closely related protein U2B" demonstrated that addition of a competitor RNA can suppress selection of a protein with a higher affinity for a given RNA target, thereby allowing the preferential amplification of a lower affinity protein. Together, these findings suggest that T7 phage display can be used to rapidly and selectively clone virtually any protein that binds a known RNA regulatory element, including those that bind with low affinity or that must compete for binding with other proteins.

Bacteriophage T7↗

PubMed2001

Effect of genetic selection for group productivity and longevity on immunological and hematological parameters of chickens.

A line of White Leghorn chickens was selected for high group productivity and longevity resulting in improved survival and feather score as well as reduced cannibalism and flightiness. Improvements in survival might have also been due to improved immunity. The present study was designed to test the hypothesis that selection for high (HGPS) and low (LGPS) group productivity and survivability also altered immune and hematological parameters. The LGPS line was an intense reverse selected line of the HGPS line at the eighth generation of development. Hens were randomly assigned to individual cages at 17 wk of age. Blood samples were collected from the hens at 21 wk of age. Subsets of T lymphocytes (CD4+, CD8+, and gammadelta cells) were measured using flow cytometry. Concentrations of plasma IgG were quantified with western blot analysis and immunoprecipitation assay. Hematological parameters were collected from blood smears. The HGPS hens had significantly higher percentages of blood lymphocytes and CD4+:CD8+ ratios of circulating T cells (P < 0.01) and tended to have more, but not significantly, gammadelta T cells (P = 0.07) than the LGPS hens. In contrast, the LGPS hens exhibited eosinophilia and heterophilia and greater heterophil:lymphocyte ratios (P < 0.01). The concentrations of plasma IgG were also significantly higher in the LGPS hens (P < 0.01). These results suggest that genetic selection for group productivity and longevity also alters the immunological and hematological systems of hens. The line difference in regulation of T cells, leukocytes, and production of IgG may suggest that different genes or modes of gene action are involved.

Animals↗

PubMed1985

Circulating antibody-antigen complexes following Trypanosoma musculi infection in mice genetically selected to produce high or low affinity antibody.

Mice from lines genetically selected for the production of either high or low affinity antibody to protein antigens and which differ in their susceptibility to chronic immune complex disease were infected with Trypanosoma musculi. The parasite became patent in both lines by day 5 and no significant differences in the levels of parasites during the infection were observed between the two lines. Serum levels of both antigen non-specific and T. musculi antigen specific immune complexes were determined during the infection by the solid phase conglutinin and Clq binding assays. In both lines, antigen non-specific complexes were detected by day 15 after infection with maximum levels observed by day 30. At this time, low affinity line mice had significantly higher levels than did high line mice as determined by the Clq assay but not by the conglutinin assay. The deposition of immune complex like material in glomeruli, assessed by immunofluorescence, was associated with the clearance of the parasite and the presence of circulating antigen specific complexes. The pattern of localization of complexes in both lines was predominantly mesangial with some deposition in the capillaries. The intensity of fluorescence increased during the infection. Initially (day 10) only IgM was observed in the glomeruli but IgG1 and IgG2b were detected from day 20 to day 40. IgG2a was only detected on day 40. However, in none of the animals was this deposition of complexes associated with proteinuria. Hence, the data presented here show that the low affinity line mice produce higher levels of smaller circulating complexes following T. musculi infection than do high affinity mice. However, this does not result in significant differences in localization and induction of renal disease as seen following chronic antigen injection.

Animals↗

PubMed1991

[Miracidial attraction by Biomphalaria tenagophila genetically selected for susceptibility].

The attraction of Schistosoma mansoni miracidea by snails genetically selected for susceptibility to infection is considered. Miracidea were exposed to snails or to snail conditioned water in a specially devised three-chamber glass apparatus. Ten miracidea were used for each test and were deposited in the middle chamber (a passage); after 15 minutes, them in each of the number of the three chambers were recorded. In one of the chambers, chosen randomly at each trial, either snails or SCW were used. The snails and the SCW were chosen from either selected snails (highly susceptible) or unselected. The miracidea were of two groups: they came either from susceptible snails in previous generation or were of unselected traits. The results show that only snail generation has importance for the attraction: selected molluscs of susceptible traits do attract the miracidea more.

Animals↗