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At least 451 records · Page 25Linked to original sources
PubMed2000

The tkNeo gene, but not the pgkPuro gene, can influence the ability of the beta-globin LCR to enhance and confer position-independent expression onto the beta-globin gene.

Whether drug-selectable genes can influence expression of the beta-globin gene linked to its LCR was assessed here. With the tkNeo gene placed in cis and used to select transfected cells, the beta-globin gene was expressed fourfold lower when it was positioned upstream of the LCR rather than downstream. This difference did not occur when the pgkPuro gene replaced tkNeo. Moreover, the beta-globin gene situated upstream of the LCR was transcribed without position effects when it was cotransfected with a pgkPuro-containing plasmid, whereas cotransfection with a tkNeo plasmid gave measurable position effects. Previous results from transfected cells selected via a linked tkNeo gene suggested that the 3' end of the beta-globin gene has no impact on LCR-enhanced expression. Here, removal of the 3' end of the beta-globin gene resulted in lower and much more variable expression in both transgenic mice and cells cotransfected with pgkPuro. Together, the results suggest that tkNeo, but not pgkPuro, can strongly influence expression of the beta-globin gene linked to its LCR. The findings could partly explain why data on beta-globin gene regulation obtained from transfected cells have often not agreed with those obtained using transgenic mice. Hence, one must be careful in choosing a drug-selectable gene for cell transfection studies.

Animals↗
PubMed2003

Hematopoietic stem cell gene therapy: selecting only the best.

Hematopoietic stem cell (HSC) gene therapy can potentially cure a variety of human hematopoietic diseases, such as sickle cell disease. Selection and expansion of gene-corrected HSCs has now been accomplished for the first time using HSC from large animals - dogs and humans - with a novel drug-resistance gene, MGMT, which is not expressed in normal HSCs (see the related articles beginning on pages 1561 and 1581). Highly efficient lentiviral transfer and expression of MGMT into relatively few HSCs led to repopulation of most of the hematopoietic compartment with gene-corrected cells following suitable drug treatment. This selection system may be useful in human clinical trials to permit gene therapy in autologous and allogeneic bone marrow transplantation settings.

Animals↗
PubMed2006

Differential expression of selected mitochondrial genes in hibernating little brown bats, Myotis lucifugus.

High rates of non-shivering thermogenesis by brown adipose tissue accompanied by additional shivering thermogenesis in skeletal muscle provide the powerful reheating of body organs that allows hibernating mammals to return from their state of cold torpor back to euthermic function. Previous studies have suggested that changes to brown adipose mitochondria occur during hibernation and are partially responsible for its capacity for non-shivering thermogenesis. The current study shows that selected mitochondrial enzyme activities are elevated and selected genes and proteins are induced during torpor in brown adipose tissue of the little brown bat, Myotis lucifugus. Cytochrome oxidase activity in brown adipose tissue was more than 3-fold higher during torpor than in euthermic animals. Transcript levels of mitochondria-encoded genes, coxII and nad4, were also 3-4-fold higher during torpor, as evidenced by northern blotting. By contrast, transcripts of these genes were unchanged in skeletal muscle during torpor. Protein levels of carnitine palmitoyl transferase-1beta, an enzyme embedded in the outer membrane of the mitochondria that is the rate-limiting step enzyme in beta-oxidation, were also elevated by 2-fold during torpor in brown adipose but were unchanged in skeletal muscle. Cloning and sequencing of a 624 bp segment of cpt-1beta revealed a number of amino acid substitutions in the bat protein as compared to CPT-1beta from other mammals; these may be beneficial for enzyme function at low body temperatures during torpor. This study provides further evidence for a key role of mitochondria in hibernation.

Adipose Tissue, Brown↗
PubMed2002

Selection and gene duplication: a view from the genome.

Immediately after a gene duplication event, the duplicate genes have redundant functions. Is natural selection therefore completely relaxed after duplication? Does one gene evolve more rapidly than the other? Several recent genome-wide studies have suggested that duplicate genes are always under purifying selection and do not always evolve at the same rate.

Animals↗
PubMed2003

Evaluation of hypothalamic gene expression in mice divergently selected for heat loss.

Mouse lines divergently selected for heat loss were evaluated for correlated responses in the hypothalamic transcriptome. High (MH) heat loss mice have approximately 50% greater heat loss, approximately 35% less body fat, approximately 20% greater feed intake, approximately 100% greater locomotor activity levels, and higher core body temperature compared with low (ML) heat loss mice. We evaluated hypothalamic expression between inbred lines derived from MH and ML lines (IH and IL, respectively) using cDNA microarrays and selected genes previously isolated in a large differential-display PCR experiment. Northern analysis was used to confirm differences, revealing higher hypothalamic mRNA expression of oxytocin (Oxt) and tissue inhibitor of metalloproteinase 2 (Timp-2) in the IH line. Real-time PCR assays were developed for Oxt, Timp-2, and ribosomal protein L3 (Rpl3, previously found to be upregulated in IL) and confirmed differential expression of these genes with potential physiological relevance in energy balance. These results provide information on correlated responses in the transcriptome of mice selected for high and low energy expenditure and reveal new information regarding genetic regulation of energy balance.

Animals↗
PubMed2000

Development of a chromosome-plasmid balanced lethal system for Lactobacillus acidophilus with thyA gene as selective marker.

A chromosome-plasmid balanced lethal gene delivery system for Lactobacillus acidophilus based on the thyA gene was developed. The selected L. acidophilus DOM La strain carries a mutated thyA gene and has an obligate requirement for thymidine. This strain can be used as a host for the constructed shuttle vector pFXL03, lacking antibiotic-resistant markers but having the wild-type thyA gene from L. casei which complements the thyA chromosomal mutation. The vector also contains the replicon region from plasmid pUC19 and that of the Lactococcus plasmid pWV01, which allows the transfer between Escherichia coli, L. casei and L. acidophilus. Eight unique restriction sites (i.e., PstI, HindIII, SphI, SalI, AccI, XbaI, KpnI and SacI) are available for cloning. After 40-time transfers in modified MRS medium, no plasmid loss was observed. The vector pFXL03 is potentially useful as a food-grade vaccine delivery system for L. acidophilus.

Drug Delivery Systems↗
PubMed2006

Classification of intramural metastases and lymph node metastases of esophageal cancer from gene expression based on boosting and projective adaptive resonance theory.

Esophageal cancer is a well-known cancer with poorer prognosis than other cancers. An optimal and individualized treatment protocol based on accurate diagnosis is urgently needed to improve the treatment of cancer patients. For this purpose, it is important to develop a sophisticated algorithm that can manage a large amount of data, such as gene expression data from DNA microarrays, for optimal and individualized diagnosis. Marker gene selection is essential in the analysis of gene expression data. We have already developed a combination method of the use of the projective adaptive resonance theory and that of a boosted fuzzy classifier with the SWEEP operator denoted PART-BFCS. This method is superior to other methods, and has four features, namely fast calculation, accurate prediction, reliable prediction, and rule extraction. In this study, we applied this method to analyze microarray data obtained from esophageal cancer patients. A combination method of PART-BFCS and the U-test was also investigated. It was necessary to use a specific type of BFCS, namely, BFCS-1,2, because the esophageal cancer data were very complexity. PART-BFCS and PART-BFCS with the U-test models showed higher performances than two conventional methods, namely, k-nearest neighbor (kNN) and weighted voting (WV). The genes including CDK6 could be found by our methods and excellent IF-THEN rules could be extracted. The genes selected in this study have a high potential as new diagnosis markers for esophageal cancer. These results indicate that the new methods can be used in marker gene selection for the diagnosis of cancer patients.

Algorithms↗
PubMed1995

High-affinity triple helix formation by synthetic oligonucleotides at a site within a selectable mammalian gene.

Specific recognition of duplex DNA by a single-stranded oligonucleotide via the formation of triplex DNA is a rational approach for targeting specific regions of a genome. By screening a number of potential target sites for triple helix formation within mammalian genes that allow genetic selection in cell culture, we have identified a site within intron 1 of the hamster adenine phosphoribosyltransferase (APRT) gene that specifically binds a triplex-forming oligodeoxyribonucleotide (TFO) with high affinity. Under optimal conditions for triplex formation, the equilibrium dissociation constant is in the nanomolar range (Kd = 7 x 10(-10) M). This high-affinity binding is very specific, as a 10(5)-fold excess of genomic DNA reduced triplex formation less than 10-fold, and within a 6928-bp plasmid bearing the APRT gene, only restriction fragments containing the intron 1 site were found to bind the TFO. Results of DNase I protection assays were consistent with the TFO binding in an antiparallel orientation via reverse Hoogsteen hydrogen bonds in the major groove of the duplex. We have examined the kinetics of triplex formation as well as the effects of ionic composition and chemical modifications of the TFO on triplex formation. While divalent cations were not required for triplex formation, Mg2+ stabilized the triplex apparently through inhibition of TFO dissociation, with a mean bound lifetime of > 17 h for the triplex at Mg2+ concentrations above 5 mM.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenine Phosphoribosyltransferase↗
PubMed1997

Development of a novel selective amplifier gene for controllable expansion of transduced hematopoietic cells.

To overcome the low efficiency of gene transfer into hematopoietic cells, we developed a novel system for selective expansion of transduced cells. To this end, we constructed a chimeric cDNA (GCRER) encoding the fusion protein between the granulocyte colony-stimulating factor receptor (G-CSFR) and the hormone-binding domain (HBD) of the estrogen receptor (ER) as a selective amplifier gene. Use of the intracellular signaling pathway of G-CSFR was considered to be appropriate, because G-CSF has the ability not only to stimulate the neutrophil production, but also to expand the hematopoietic stem/progenitor cell pool in vivo. To activate the exogenous G-CSFR signal domain selectively, the estrogen/ER-HBD system was used as a molecular switch in this study. When the GCRER gene was expressed in the interleukin-3 (IL-3)-dependent murine cell line, Ba/F3, the cells showed IL-3-independent growth in response to G-CSF or estrogen. Moreover, the Ba/F3 cells transfected with the Delta(5-195)GCRER, whose product lacks the extracellular G-CSF-binding domain, did not respond to G-CSF, but retained the ability for estrogen-dependent growth. Further, murine bone marrow cells transduced with the GCRER or Delta(5-195)GCRER gene with retroviral vectors formed a significant number of colonies in response to estrogen, as well as G-CSF, whereas estrogen did not stimulate colony formation by untransduced murine bone marrow cells. It is noteworthy that erythroid colonies were apparently formed by the bone marrow cells transduced with the GCRER gene in the presence of estrogen without the addition of erythropoietin, suggesting that the signals from the G-CSFR portion of the chimeric molecules do not preferentially induce neutrophilic differentiation, but just promote the differentiation depending on the nature of the target cells. We speculate that when the selective amplifier genes are expressed in the primitive hematopoietic stem cells, the growth signal predominates and that the population of transduced stem cells expands upon estrogen treatment, even if some of the cells enter the differentiation pathway. The present study suggests that this strategy is applicable to the in vivo selective expansion of transduced hematopoietic stem cells.

Animals↗
PubMed1992

Selective expression of trypsin fusion genes in acinar cells of the pancreas and stomach of transgenic mice.

Fusion genes combining the 5'-transcriptional regulatory region of the rat trypsin I gene and the structural gene of human growth hormone as a reporter were expressed to the high levels characteristic of the endogenous trypsin I gene selectively in the acinar cells of the pancreas of transgenic mice. As little as 232 base pairs of trypsin gene sequences containing the transcriptional start site and upstream promoter elements were sufficient to direct pancreatic expression. The tissue-specific expression was controlled transcriptionally. Trypsin-human growth hormone fusion transgenes also were expressed, although at low levels, in the stomach, an unexpected site for the expression of pancreatic digestive enzymes. Expression in the stomach of endogenous trypsin, elastase, and amylase genes in both normal and transgenic mice verified that transgene expression was consistent with normal expression of pancreatic genes. Endogenous amylase colocalizes with pepsinogen in the acinar cell-like Chief cells of the glandular portion of the mouse stomach. The expression of pancreatic genes in stomach cells is probably the consequence of similar developmental origins of pancreatic and gastric acinar cells from the primordial gut.

Animals↗
PubMed2003

Tumor cell-collagen interactions: Identification and semi-quantitative evaluation of selectively-expressed genes by combination of differential display- and multiplex-PCR.

It is widely acknowledged that the presence of extracellular matrix components as substrates can drastically modulate the phenotype and gene expression of cultured cells, including tumor cells. A number of published reports indicated that substrates made from two peculiar collagen species, i.e. type V and OF/LB, which are abnormally deposited in the stroma of primary ductal infiltrating carcinoma (d.i.c.) of the breast "in vivo," were able to exert marked and opposite effects on "in vitro" viability, growth and invasiveness of the 8701-BC cell line, isolated from d.i.c.-affected breast epithelium. To complement such functional data on the effect of cell-collagen interactions with information at molecular level, we have utilized a combination of differential display- and semi-quantitative multiplex-PCR techniques with the aim of detecting variations in the expression levels of selected genes by cells maintained in either culture condition. Here we report some prototypical data on the identification and semi-quantitation of three of the differentially-amplified PCR products found, i.e. HSP2A and MSF-B which are up-regulated in cells grown onto OF/LB collagen substrate, and SRCAP which is prominently down-regulated in the presence of type V collagen substrate. This protocol represents a powerful tool for evaluating changes in the levels and patterns of gene expression which can be theoretically adapted to any experimental model system.

Journal Article↗
PubMed2003

Selection conflicts, gene expression, and codon usage trends in yeast.

Synonymous codon usage in yeast appears to be influenced by natural selection on gene expression, as well as regional variation in compositional bias. Because of the large number of potential targets of selection (i.e., most of the codons in the genome) and presumed small selection coefficients, codon usage is an excellent model for studying factors that limit the effectiveness of selection. We use factor analysis to identify major trends in codon usage for 5836 genes in Saccharomyces cerevisiae. The primary factor is strongly correlated with gene expression, consistent with the model that a subset of codons allows for more efficient translation. The secondary factor is very strongly correlated with third codon position GC content and probably reflects regional variation in compositional bias. We find that preferred codon usage decreases in the face of three potential limitations on the effectiveness of selection: reduced recombination rate, increased gene length, and reduced intergenic spacing. All three patterns are consistent with the Hill-Robertson effect (reduced effectiveness of selection among linked targets). A reduction in gene expression in closely spaced genes may also reflect selection conflicts due to antagonistic pleiotropy.

Codon↗
PubMed1999

Broad-sense sexual selection, sex gene pool evolution, and speciation.

Studies of sexual selection have traditionally focused on explaining the extreme sexual dimorphism in male secondary sexual traits and elaborate mating behaviors displayed by males during courtship. In recent years, two aspects of sexual selection have received considerable attention in the literature: an extension of the sexual selection concept to other traits (i.e., postcopulatory behaviors, external and internal genital morphology, gametes, molecules), and alternative mechanistic explanations of the sexual selection process (i.e., coevolutionary runaway, good-genes, sexual conflicts). This article focuses on the need for an extension of sexual selection as a mechanism of change for courtship and (or) mating male characters (i.e., narrow-sense sexual selection) to all components of sexuality not necessarily related to courtship or mating (i.e., broad-sense sexual selection). We bring together evidence from a wide variety of organisms to show that sex-related genes evolve at a fast rate, and discuss the potential role of broad-sense sexual selection as an alternative to models that limit speciation to strict demographic conditions or treat it simply as an epiphenomenon of adaptive evolution.

Animals↗
PubMed1990

Increased rate of base substitution in a hamster mutator strain obtained during serial selection for gene amplification.

The pattern of mutations produced by a mutator gene (obtained during serial selection for amplification of the dihydrofolate reductase [dhfr] locus) shows a pronounced shift from that found in wild-type cells. The rate of certain types of base substitutions (particularly transitions) is dramatically increased, while gene rearrangements constitute a lower proportion of mutations. These data suggest a lower fidelity of the replication process in the mutator strain.

Animals↗
PubMed1984

Cell-specified expression of a selectable hybrid gene.

We have inserted into a retroviral vector the structural information encoding the selectable bacterial genes neo and gpt linked, respectively, to the promoters of the herpesvirus thymidine kinase gene and the rat preproinsulin II gene. We have used this recombinant retrovirus construction to transduce insulinoma cell lines that are positive or negative for insulin expression. Selection of the transductants for neo yielded resistant colonies from all of the cell lines, while selection for gpt yielded resistant transductants in relatively high frequency only from insulin-producing cells. The 5' end of the gpt transcripts maps to the authentic preproinsulin capping site of the construction.

Animals↗
PubMed2006

Identification of Salmonella enterica serovar Typhimurium using specific PCR primers obtained by comparative genomics in Salmonella serovars.

Salmonella enterica serovar Typhimurium is a major foodborne pathogen throughout the world. Until now, the specific target genes for the detection and identification of serovar Typhimurium have not been developed. To determine the specific probes for serovar Typhimurium, the genes of serovar Typhimurium LT2 that were expected to be unique were selected with the BLAST (Basic Local Alignment Search Tool) program within GenBank. The selected genes were compared with 11 genomic sequences of various Salmonella serovars by BLAST. Of these selected genes, 10 were expected to be specific to serovar Typhimurium and were not related to virulence factor genes of Salmonella pathogenicity island or to genes of the O and H antigens of Salmonella. Primers for the 10 selected genes were constructed, and PCRs were evaluated with various genomic DNAs of Salmonella and non-Salmonella strains for the specific identification of Salmonella serovar Typhimurium. Among all the primer sets for the 10 genes, STM4497 showed the highest degree of specificity to serovar Typhimurium. In this study, a specific primer set for Salmonella serovar Typhimurium was developed on the basis of the comparison of genomic sequences between Salmonella serovars and was validated with PCR. This method of comparative genomics to select target genes or sequences can be applied to the specific detection of microorganisms.

Base Sequence↗
PubMed2001

Expression of herpes simplex virus-thymidine kinase gene controlled by a promoter region of the midkine gene confers selective cytotoxicity to ganciclovir in human carcinoma cells.

A selective expression of suicide gene(s) in tumor cells should produce a preferential cytotoxic effect on tumors. Promoter region(s) of a gene that is expressed in tumors but not in normal tissues can be useful for tumor-specific transcription of a suicide gene. Midkine (MK), a growth/differentiation factor, is expressed predominantly in various types of human tumors, whereas its expression in adult normal tissues is highly restricted. In our study, we showed that a 2.3-kb fragment of genomic DNA in the 5' upstream region of the MK gene could activate transcription of a fused reporter gene in MK-positive cells but not in MK-negative cells. Efficiency of the cis-acting sequence to permit expression of an exogenous gene in tumor cells was comparable with that of the SV40 promoter. Regulated expression of the herpes simplex virus-thymidine kinase (HSV-TK) gene under the control of the MK promoter conferred increased sensitivity to ganciclovir (GCV) on MK-positive tumor cells. Administration of GCV into nude mice that were implanted with MK-positive tumor cells that expressed the HSV-TK gene under the control of the MK promoter could suppress the subsequent tumor growth. Expression of therapeutic genes restricted to tumors can be achieved by the use of the putative cis-acting MK promoter.

Animals↗
PubMed2003

Neural and orofacial defects in Folp1 knockout mice [corrected].

BACKGROUND: Folic acid is essential for the development of the nervous system and other associated structures. Mice deficient in the folic acid-binding protein one (Folbp1) gene display multiple developmental abnormalities, including neural and craniofacial defects. To better understand potential interactions between Folbp1 gene and selected genes involved in neural and craniofacial morphogenesis, we evaluated the expression patterns of a panel of crucial differentiation markers (Pax-3, En-2, Hox-a1, Shh, Bmp-4, Wnt-1, and Pax-1). METHODS: Folbp1 mice were supplemented with low dosages of folinic add to rescue nullizygotes from dying in utero before gestational day 10. The gene marker analyses were carried out by in situ hybridization. RESULTS: In nullizygote embryos with open cranial neural tube defects, the downregulation of Pax-3 and En-2 in the impaired midbrain, along with an observed upregulation of the ventralizing marker Shh in the expanded floor plate, suggested an important regulatory interaction among these three genes. Moreover, the nullizygotes also exhibit craniofacial abnormalities, such as cleft lip and palate. Pax-3 signals in the impaired medial nasal primordia were significantly increased, whereas Pax-1 showed no expression in the undeveloped lateral nasal processes. Although Shh was downregulated, Bmp-4 was strongly expressed in the medial and lateral nasal processes, highlighting the antagonistic activities of these molecules. CONCLUSIONS: Impairment of Folbp1 gene function adversely impacts the expression of several critical signaling molecules. Mis-expression of these molecules, perhaps mediated by Shh, may potentially contribute to the observed failure of neural tube closure and the development of craniofacial defects in the mutant mice.

Animals↗