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PubMed2003

[Study on the preparing of polyclonal antibodies against plant-selected maker gene hpt expression protein by DNA immunization].

AIM: To prepare the polyclonal antibodies (pAbs) against HPT, a kind of plant-selected maker gene encoding protein, by DNA immunization technique and explore the influencing factors for this gene immunization. METHODS: The coding sequence of hpt was cloned into eukaryotic expression vector pCDNA3. The sequence of the plasmid pCDNA3-HPT was demonstrated by restricting enzyme digestion analysis and DNA sequencing. The sequence-correct recombinant plasmids were purified and used to immunize BALB/c mice. The titer and specificity of antisera were detected by ELISA and Western blot, respecitively. RESULTS: No pAb against HPT was detected following three immunization with hpt genes. Then mice were divided into three groups when the forth booster immunization was carried out: 1st group (immunization with the endotoxin-free recombinant plasmids), 2nd group (immunization with (His)(6)-HPT fusion protein expressed in E.coli) and 3rd group (immunization with the same plasmids as before mentioned). As a result, the pAb titer of the 1st group mice increased to 1:200, and the that of 2nd group was up to 1:2 000. Yet the 3rd group detected no anti-HPT antibody. Western blot analysis had proved that antisera of the first two groups could produce specific binding reaction to the purified GST-HPT, (His)(6)-HPT protein and their expressed product (bacterial protein). CONCLUSION: We have got successfully the specific pAb against HPT by DNA immunization, but its titer is yet unsatisfactory, inferring that the character of the hpt gene self and its expression level may play an important role. In order to raise level of serum antibody prepared by DNA immunization, the farther study on various influencing factors still need to be performed.

Animals↗
PubMed1998

Co-integration, co-expression and co-segregation of an unlinked selectable marker gene and NtFAD3 gene in transgenic rice plants produced by particle bombardment.

Using the scutellar tissue of rice mature embryos as a target tissue, a selectable marker gene, bar, and an unselectable gene, fatty acid desaturase gene from tobacco (NtFAD3), on separate plasmids, were introduced by particle bombardment. Co-integration, co-expression and inheritance of these genes were analyzed as well as seed fertility of the transgenic plants. Twenty-three out of 32 bialaphos-resistant plants integrated the NtFAD3 gene, which was confirmed by Southern-blot analysis of R0 plants, and showed one to more than 20 hybridizing bands of exogenous DNA, indicating a 72% (23/32) co-integration frequency. However, the frequency of the transgenic plants containing the 1.4-kb fragment of NtFAD3 gene was 34% (11/32). Northern-blot analysis revealed that seven out of ten fertile transgenic rice plants which had a 1.4-kb fragment of NtFAD3 cDNA expressed NtFAD3 mRNA. The NtFAD3 gene under the control of CaMV35S promoter stably expressed in the transgenic rice plants and modified the proportions of linoleic acid (18:2) and linolenic acid (18:3) in fatty acids; the content of 18:2 decreased and that of 18:3 increased. Fourteen out of 32 (44%) transgenic plants set seeds and 18 (56%) showed low fertility or sterility. Molecular analysis of the selfed progeny indicated that all copies in almost all R0 plants were inherited as a single dominant hemizygous locus.

Fatty Acid Desaturases↗
PubMed2000

Latitude-correlated genetic polymorphisms: selection or gene flow?

Latitude-correlated polymorphisms can be due to either selection-driven evolution or gene flow. To discriminate between them, we propose an approach that studies subpopulations springing from a single population that have lived for generations at different latitudes and have had a low genetic admixture. These requirements are fulfilled to a large extent by Ashkenazi and Sephardi Jews. The original population lived at a latitude of 35 degrees N, where the Sephardis still live. The Ashkenazis, however, moved to a latitude of 50 degrees N, starting about 10 centuries ago. The present study examines 3 latitude-correlated polymorphisms: PGP, PGM1, and AHSG. We found that PGP*2 and AHSG*2 alleles most likely underwent selection-driven evolution, but that PGM1*ts allele was not similarly affected. Since temperature might have been considered a reasonable selective factor, we also studied a population living at >800 m above sea level from Aosta Valley (Italy).

Altitude↗
PubMed2006

Incorporating genetic analyses into birth defects cluster investigations: strategies for identifying candidate genes.

BACKGROUND: Incorporating genetic analyses into birth defect cluster investigations may increase understanding of both genetic and environmental risk factors for the defect. Current constraints of most birth defect cluster investigations make candidate gene selection the most feasible approach. Here, we describe strategies for choosing candidate genes for such investigations, which will also be applicable to more general gene-environment studies. METHODS: We reviewed publicly available web-based resources for selection of candidate genes and identification of risk factors, as well as publications on different strategies for candidate gene selection. RESULTS: Candidate gene selection requires consideration of available gene-disease databases, previous epidemiological studies, animal model research, linkage and expression studies, and other resources. We describe general considerations for utilizing available resources, as well as provide an example of a search for candidate genes related to gastroschisis. CONCLUSIONS: Available web resources could facilitate selection of candidate genes, but selection of optimal candidates will still require a strong understanding of genetics and the pathogenesis of the defect, as well as careful consideration of previous epidemiological studies.

Animals↗
PubMed1995

Selective inhibition of repair of active genes by hyperthermia is due to inhibition of global and transcription coupled repair pathways.

Hyperthermia specifically inhibits the repair of UV-induced DNA photolesions in transcriptionally active genes. To define more precisely which mechanisms underlie the heat-induced inhibition of repair of active genes, removal of cyclobutane pyrimidine dimers (CPDs) was studied in human fibroblasts with different repair capacities and different transcriptional status of the adenosine deaminase gene, i.e. normal human cells, human cells carrying an inactive copy of the adenosine deaminase gene and xeroderma pigmentosum complementation group C fibroblasts. The results indicate that repair of active genes is impaired by inhibition of two repair pathways: (i) a global repair system involved in the repair of CPDs in potentially active genes; and (ii) the transcription-coupled repair pathway responsible for the accelerated repair of the transcribed strand. Since X-ray-induced DNA damage is also preferentially removed from the transcribed strand of active genes, selective inhibition of repair of radiation-induced DNA damage in active genes may play a key role in radiosensitization due to hyperthermia.

Adenosine Deaminase↗
PubMed2005

Identification of global gene expression differences between human lens epithelial and cortical fiber cells reveals specific genes and their associated pathways important for specialized lens cell functions.

PURPOSE: In order to identify specific genes that may play important roles in maintaining the specialized functions of lens epithelial and fiber cells, we have analyzed the global gene expression profiles of these two cell types in the human lens. This analysis will also reveal those genes that are exclusively expressed in the epithelial and cortical fiber cells and those genes that may play important roles in the differentiation of epithelial cells to mature fiber cells. METHODS: Oligonucleotide microarray hybridization was used to analyze the expression profiles of 22,215 genes between adult (average age greater than 56 years) human lens epithelial and cortical fiber cells. The expression levels of selected genes were further compared by semi-quantitative RT-PCR and selected genes were functionally clustered into common categories using the EASE bioinformatics software package. RESULTS: Analysis of three separate microarray hybridizations revealed 1,196 transcripts that exhibit increased expression and 1,278 transcripts that exhibit decreased expression at the 2 fold or greater level between lens epithelial cells and cortical fiber cells on all three of the arrays analyzed. Of these, 222 transcripts exhibited increased expression and 135 transcripts exhibited decreased expression by an average of 5 fold or greater levels on all three arrays. Semi-quantitative RT-PCR analysis of 21 randomly selected genes revealed identical expression patterns as those detected by microarray hybridization indicating that the microarray data are accurate. Functional clustering of the identified gene expression patterns using the EASE program revealed a wide variety of biological pathways that exhibited altered expression patterns between the two cell types including mRNA processing, cell adhesion, cell proliferation, translation, protein folding, oxidative phosphorylation, and apoptosis, among others. CONCLUSIONS: These data reveal novel and previously identified gene expression differences between lens epithelial and cortical fiber cells. The gene expression differences indicate distinct pathways and functions important for the specialization of lens epithelial and fiber cells and provide insight into potential mechanisms important for lens cell differentiation.

Aged↗
PubMed2006

Local adaptation, patterns of selection, and gene flow in the Californian serpentine sunflower (Helianthus exilis).

The traditional view of the species as the fundamental unit of evolution has been challenged by observations that in heterogeneous environments, gene flow may be too restricted to overcome the effects of local selection. Whether a species evolves as a cohesive unit depends critically on the dynamic balance between homogenizing gene flow among populations and potentially disruptive local adaptation. To examine this evolutionary balance between "global" gene flow and local selection, we studied northern Californian populations of Helianthus exilis, the serpentine sunflower, within a mosaic of contrasting serpentine and nonserpentine areas that differ considerably in soil chemistry and water availability. Local adaptation to riparian and serpentine habitats was studied in Helianthus exilis along with an analysis of gene flow patterns among populations within these habitats. Local adaptation was assessed in H. exilis during 2002 and 2003 using reciprocal transplant experiments at multiple locations within serpentine and riparian habitats. Effects of competition and germination date on the expression of local adaptation were also examined within the reciprocal transplant experiments. Local adaptation was detected in both years at the local site level and at the level of habitat. The analysis of the transplanted populations indicated that the patterns of selection differed considerably between riparian and serpentine sites. Differential survivorship occurred in serpentine habitats, whereas selection on reproductive output predominated in riparian habitats. Local adaptation was expressed only in the absence of competition. Local adaptation in terms of survivorship was most strongly expressed in treatments with delayed seed germination. Microsatellite markers were used to quantify population genetic parameters and examine the patterns of gene flow among sampled populations. Analysis of molecular markers revealed a system of population patches that freely exchange genes with each other. Strong selection seems to maintain ecotypic variation within this endemic sunflower species, while extensive gene flow among populations prevents local speciation between serpentine and riparian ecotypes.

Adaptation, Physiological↗
PubMed1991

5' regulatory region of a novel cytokine gene mediates selective activation by interferon gamma.

A newly described member of the platelet factor 4 family of cytokine genes, mig, is selectively induced by interferon gamma (IFN-gamma), and not IFN-alpha, in the mouse macrophage-like cell line RAW 264.7. Treatment of RAW 264.7 cells with IFN-gamma activated mig gene transcription as determined by nuclear run-on assays. mig genomic clones were isolated, and constructs containing genomic fragments that included the mig promoter region and the CAT reporter gene were prepared. In RAW 264.7 cells transfected with these constructs, CAT activity was found to be selectively induced by IFN-gamma. A 278-bp genomic fragment containing 235 nucleotides 5' of the transcription start site was sufficient for IFN-gamma-selective induction of CAT activity. Analysis of 5' deletion mutants localized a region essential for activation by IFN-gamma to within 64 nucleotides extending from -235 to -172. A genomic fragment containing this sequence was capable of conferring IFN-gamma inducibility to constructs with a heterologous promoter.

Animals↗
PubMedDate not supplied

Mechanisms and applications of DNA-mediated gene transfer in mammalian cells - a review.

The ability of mammalian cells to take up exogenously added DNA and to express genes included on that DNA has been well documented. DNA-mediated gene transfer (DMGT) potentially is a useful technique for the elucidation of many of the factors that control gene expression, and for the purification and isolation of mammalian genes. Before many of the benefits can be realized, however, a more detailed understanding of the organization, intracellular location, and expression of transferred genes will be needed. Recent studies have begun to characterize the DMGT process. Selected genes become linked to other exogenously added DNA during or subsequent to transfer and persist in the nuclei of recipient cells as part of large molecules called transgenomes. Transgenomes initially are maintained unstably and are lost from the population with first order kinetics. After a variable number of generations in culture, subpopulations arise that maintain the transferred genes stably. In these "stable" cells the transgenome is associated with a recipient cell chromosome, although the particular chromosome differs in independent "stable" lines. Mixture of an excess of specific nonselectable genes with the selected gene prior to transfer results in the inclusion of the nonselected genes in the transgenomes present in most cells that survive selection. This finding demonstrates the feasibility of introducing virtually any purified gene into mammalian cells. Recently microinjection of DNA directly into the nuclei of cells has been demonstrated. This technique greatly increases the frequency of gene transfer and significantly expands the number of cell types that can be genetically transformed.

Animals↗
PubMed2003

Improved gene transfer selectivity to hepatocarcinoma cells by retrovirus vector displaying single-chain variable fragment antibody against c-Met.

Engineered retroviruses are widely used vectors for cancer gene therapy approaches. However, the ability to target cells of therapeutic interest while controlling the expression of the transferred genes would improve both the efficiency and the safety of viral vectors. In this study, we investigated the ability of a retroviral amphotropic envelope displaying single-chain variable-fragment (scFv) directed against the c-Met receptor, to target the entry of recombinant retroviruses to human hepatocarcinoma cells. Four single-chain antibody fragments directed against the c-Met receptor were generated and inserted into the viral envelope protein as an N-terminal fusion. The modified envelopes were incorporated into virus particles and one of the chimeric viruses, 3D6-Env, transduced preferentially human hepatoma cells rather than proliferating human hepatocytes. In another construct, the urokinase cleavage site was inserted between the scFv moiety and the envelope. Chimeric scFv-urokinase-Env viruses transduced hepatoma cells with a similar efficiency to that of the control virus and their infectivity in human hepatocytes remained low. These results indicate that amphotropic retroviruses with engineered envelopes to display scFv directed against the c-Met receptor can efficiently and selectively deliver genes into hepatoma cells.

Antibodies↗
PubMed2003

Bifidobacterium adolescentis as a delivery system of endostatin for cancer gene therapy: selective inhibitor of angiogenesis and hypoxic tumor growth.

In order to overcome difficulties that hampered widespread application of antiangiogenesis in cancer therapy, a highly specific delivery system may be engaged in vivo to deliver and express antiangiogenic genes. We selected a strain of Bifidobacterium adolescentis (B. adolescentis) as the delivery system to transport endostatin gene to solid tumors. B. adolescentis with endostatin gene were injected into tumor-bearing mice through the tail vein. After the mice were sacrificed, the tumor and some normal tissues of the mice were examined. B. adolescentis were only found in the tumors and no bacilli were found in other normal tissues. Also, a strong inhibition of angiogenesis had been shown to inhibit local tumor growth in the administrated group. These results suggested that B. adolescentis only germinated and proliferated in solid tumors and might be a highly specific and efficient vector for transporting anticancer genes into target tumor in cancer gene therapy.

Angiogenesis Inhibitors↗
PubMed2002

Expression profiling to predict outcome in breast cancer: the influence of sample selection.

Gene expression profiling of tumors using DNA microarrays is a promising method for predicting prognosis and treatment response in cancer patients. It was recently reported that expression profiles of sporadic breast cancers could be used to predict disease recurrence better than currently available clinical and histopathological prognostic factors. Having observed an overlap in those data between the genes that predict outcome and those that predict estrogen receptor-alpha status, we examined their predictive power in an independent data set. We conclude that it may be important to define prognostic expression profiles separately for estrogen receptor-alpha-positive and estrogen receptor-alpha-negative tumors.

Breast Neoplasms↗
PubMed2003

SamCluster: an integrated scheme for automatic discovery of sample classes using gene expression profile.

MOTIVATION: Feature (gene) selection can dramatically improve the accuracy of gene expression profile based sample class prediction. Many statistical methods for feature (gene) selection such as stepwise optimization and Monte Carlo simulation have been developed for tissue sample classification. In contrast to class prediction, few statistical and computational methods for feature selection have been applied to clustering algorithms for pattern discovery. RESULTS: An integrated scheme and corresponding program SamCluster for automatic discovery of sample classes based on gene expression profile is presented in this report. The scheme incorporates the feature selection algorithms based on the calculation of CV (coefficient of variation) and t-test into hierarchical clustering and proceeds as follows. At first, the genes with their CV greater than the pre-specified threshold are selected for cluster analysis, which results in two putative sample classes. Then, significantly differentially expressed genes in the two putative sample classes with p-values < or = 0.01, 0.05, or 0.1 from t-test are selected for further cluster analysis. The above processes were iterated until the two stable sample classes were found. Finally, the consensus sample classes are constructed from the putative classes that are derived from the different CV thresholds, and the best putative sample classes that have the minimum distance between the consensus classes and the putative classes are identified. To evaluate the performance of the feature selection for cluster analysis, the proposed scheme was applied to four expression datasets COLON, LEUKEMIA72, LEUKEMIA38, and OVARIAN. The results show that there are only 5, 1, 0, and 0 samples that have been misclassified, respectively. We conclude that the proposed scheme, SamCluster, is an efficient method for discovery of sample classes using gene expression profile. AVAILABILITY: The related program SamCluster is available upon request or from the web page http://www.sph.uth.tmc.edu:8052/hgc/Downloads.asp.

Algorithms↗
PubMed2006

Natural selection on gene expression.

Changes in genetic regulation contribute to adaptations in natural populations and influence susceptibility to human diseases. Despite their potential phenotypic importance, the selective pressures acting on regulatory processes in general and gene expression levels in particular are largely unknown. Studies in model organisms suggest that the expression levels of most genes evolve under stabilizing selection, although a few are consistent with adaptive evolution. However, it has been proposed that gene expression levels in primates evolve largely in the absence of selective constraints. In this article, we discuss the microarray-based observations that led to these disparate interpretations. We conclude that in both primates and model organisms, stabilizing selection is likely to be the dominant mode of gene expression evolution. An important implication is that mutations affecting gene expression will often be deleterious and might underlie many human diseases.

Animals↗
PubMed1995

Expression of foreign genes and selection of promoter sequences in Acholeplasma laidlawii.

The stable maintenance and expression of foreign genes in mollicutes (mycoplasmas) have been difficult to achieve due to the lack of suitable vectors. In this paper we show for the first time that a replicating vector can been used to express foreign genes other than antibiotic resistance genes in Acholeplasma laidlawii. Plasmids derived from the lactococcal vector pNZ18 could introduce and maintain four different genes for many generations in A. laidlawii. One of these, encoding the dominant membrane lipoprotein spiralin from the mollicute Spiroplasma citri, was expressed; however, expression was weak, the signal peptide of spiralin was not cleaved and the protein was not covalently modified by fatty acids. This resulted in a hydrophilic character of spiralin and its cytoplasmic localization in A. laidlawii. To increase the expression of foreign genes, random A. laidlawii DNA fragments were cloned into a pNZ18-related plasmid and expression signals were selected using the Bacillus licheniformis alpha-amylase gene as a probe. Selection was done in Escherichia coli as well as directly in A. laidlawii. Active recombinants from E. coli were also able to express alpha-amylase activities and an enzyme of native size in A. laidlawii. The highest activity was obtained from a recombinant selected directly in A. laidlawii. This is the first example of a promoter sequence selected in a mollicute. Analysis of the putative promoters in seven clones revealed similar -10 and -35 regions, and similar spacer distances in A. laidlawii, Acholeplasma oculi, Lactococcus and E. coli. Vectors related to pNZ18 should be useful for the genetic analysis of specific A. laidlawii proteins and functions.

Acholeplasma laidlawii↗
PubMed2004

Joint analysis of two microarray gene-expression data sets to select lung adenocarcinoma marker genes.

BACKGROUND: Due to the high cost and low reproducibility of many microarray experiments, it is not surprising to find a limited number of patient samples in each study, and very few common identified marker genes among different studies involving patients with the same disease. Therefore, it is of great interest and challenge to merge data sets from multiple studies to increase the sample size, which may in turn increase the power of statistical inferences. In this study, we combined two lung cancer studies using microarray GeneChip, employed two gene shaving methods and a two-step survival test to identify genes with expression patterns that can distinguish diseased from normal samples, and to indicate patient survival, respectively. RESULTS: In addition to common data transformation and normalization procedures, we applied a distribution transformation method to integrate the two data sets. Gene shaving (GS) methods based on Random Forests (RF) and Fisher's Linear Discrimination (FLD) were then applied separately to the joint data set for cancer gene selection. The two methods discovered 13 and 10 marker genes (5 in common), respectively, with expression patterns differentiating diseased from normal samples. Among these marker genes, 8 and 7 were found to be cancer-related in other published reports. Furthermore, based on these marker genes, the classifiers we built from one data set predicted the other data set with more than 98% accuracy. Using the univariate Cox proportional hazard regression model, the expression patterns of 36 genes were found to be significantly correlated with patient survival (p < 0.05). Twenty-six of these 36 genes were reported as survival-related genes from the literature, including 7 known tumor-suppressor genes and 9 oncogenes. Additional principal component regression analysis further reduced the gene list from 36 to 16. CONCLUSION: This study provided a valuable method of integrating microarray data sets with different origins, and new methods of selecting a minimum number of marker genes to aid in cancer diagnosis. After careful data integration, the classification method developed from one data set can be applied to the other with high prediction accuracy.

Adenocarcinoma↗
PubMed1987

Organization of the murine Ig-related lambda 5 gene transcribed selectively in pre-B lymphocytes.

lambda 5 is an immunoglobulin lambda light chain-related gene which is selectively transcribed in murine pre-B lymphocytes to yield a 1.2 kb poly(A)+ mRNA. Comparison of the nucleotide sequence of a 1 kb cDNA clone with the sequence of a genomic clone isolated from 70Z/3 murine pre-B lymphoma cells shows lambda 5 is composed of three exons spanning a 3.75 kb DNA segment. Conserved splice signal sequences at all exon/intron boundaries and the presence of a long open reading frame indicate that a functional mRNA molecule can be made. Exon I contains a cap-site and a potential ATG start codon as well as sequences encoding a signal peptide. This gene could encode a lambda 5 protein of 209 amino acids which has, however, not yet been identified. The 3' portion of exon II and all of exon III shows strong sequence homologies to J lambda L and C lambda L exons. Homology to the lambda L chain genes is lost in the 5' portion of exon II and throughout exon I. In exon I short homologies to leader sequences and to VH framework 1 sequences are seen.

Amino Acid Sequence↗
PubMed2000

Intrachromosomal recombination between attP regions as a tool to remove selectable marker genes from tobacco transgenes.

Recombinant genes conferring resistance to antibiotics or herbicides are widely used as selectable markers in plant transformation. Once transgenic material has been selected, the marker gene is dispensable. We report a novel strategy to remove undesirable parts of a transgene after integration into the tobacco genome. This approach is based on the transfer of a vector containing a NPTII gene flanked by two 352 bp attachment P (attP) regions of bacteriophage lambda, and the identification of somatic tissue with deletion events following intrachromosomal recombination between the attP regions. This system was used to delete a 5.9 kb region from a recombinant vector that had been inserted into two different genomic regions. As the attP system does not require the expression of helper proteins to induce deletion events, or a genetic segregation step to remove recombinase genes, it should provide a useful tool to remove undesirable transgene regions, especially in vegetatively propagated species.

Bacteriophage lambda↗