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Results for “gene expression variability”

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At least 199 records · Page 11Linked to original sources
PubMed2003

Variable expression of CYP and Pgp genes in the human small intestine.

BACKGROUND: The small intestine is receiving increased attention for its importance in drug metabolism. However, knowledge of the intervariability and regulation of the enzymes involved, cytochrome p450 and P-Glycoproteins (CYP and Pgp), is poor when compared with the corresponding hepatic enzymes. METHODS: The expression of eight different CYP genes and the Pgp were determined by reverse transcription polymerase chain reaction (RT-PCR) in 51 human duodenum biopsies. And the variability and correlation of expression was analyzed. RESULTS: Extensive interindividual variability was found in the expression of most of the genes. Only CYP2C9, CYP3A4 and Pgp were found in all samples. CYP1A2, CYP2A6 and CYP2E1 exhibited the highest interindividual variability. No strong correlation of expression existed between the genes. But a highly significant correlation was found between CYP2D6/1A2, 2D6/2E1, 1A2/2E1 and 2B6/2C9. Acetylsalicylic acid and omeprazole significantly increased the expression of CYPs 2A6, 2E1 and 3A4, respectively. CONCLUSIONS: Extensive interindividual variability is characteristic for the expression of drug-metabolizing CYP and Pgp genes in human duodenum, and external factors such as drugs may further increase the variability. It is possible that the large interindividual variability may lead to variable bioavailability of orally used drugs and hence complicate optimal drug therapy, especially for drugs with a small therapeutic window. Elucidation of factors contributing to clinically important variances warrants further investigation.

Adult↗
PubMed2000

Importance of replication in microarray gene expression studies: statistical methods and evidence from repetitive cDNA hybridizations.

We present statistical methods for analyzing replicated cDNA microarray expression data and report the results of a controlled experiment. The study was conducted to investigate inherent variability in gene expression data and the extent to which replication in an experiment produces more consistent and reliable findings. We introduce a statistical model to describe the probability that mRNA is contained in the target sample tissue, converted to probe, and ultimately detected on the slide. We also introduce a method to analyze the combined data from all replicates. Of the 288 genes considered in this controlled experiment, 32 would be expected to produce strong hybridization signals because of the known presence of repetitive sequences within them. Results based on individual replicates, however, show that there are 55, 36, and 58 highly expressed genes in replicates 1, 2, and 3, respectively. On the other hand, an analysis by using the combined data from all 3 replicates reveals that only 2 of the 288 genes are incorrectly classified as expressed. Our experiment shows that any single microarray output is subject to substantial variability. By pooling data from replicates, we can provide a more reliable analysis of gene expression data. Therefore, we conclude that designing experiments with replications will greatly reduce misclassification rates. We recommend that at least three replicates be used in designing experiments by using cDNA microarrays, particularly when gene expression data from single specimens are being analyzed.

DNA, Complementary↗
PubMed2004

Variable expression of human lactoferrin gene in mice milk driven by its 90 KB upstream flanking sequences.

One major drawback in research of animal mammary gland bioreactors is the low production rate of high-expressing transgenic animals due to position effects. To obtain high and stable expression of foreign gene, yeast and bacterial artificial chromosome have been used as transgene vector in recent research. Human lactoferrin is a bioactive, versatile protein, and has large potential in nutritional and therapeutic applications. Therefore, production of recombinant lactoferrin using animal bioreactors was studied widely to satisfy its large requirement. We reported here a transgenic mice model with high-level expression of recombinant human lactoferrin in mammary gland. Transgene construct used here was a human bacterial artificial chromosome containing intact lactoferrin-encoding transcript unit, approximately 90 kb 5'-flanking sequences and 27.2 kb 3'-flanking sequences. We obtained totally 10 transgenic mice whereas two of them lacked of part of upstream sequences of the gene. Milk of eight transgenic mice line was detected by Western blot and radioimmunoassay and seven lines expressed recombinant human lactoferrin at high but variable level (0.29, 0.53, 0.90, 1.23, 2.76, 3.58, and 8.02 mg/mL, respectively). The variability of expression indicates that even the 90 kb 5' flanking sequence of the transgene can't overcome position effects completely. Moreover, we also determined sequences of 9.3 kb regulatory region and 10.6 kb encoding region of the gene and thus supplemented all unknown sequences. Our results suggested that transgene vector used here has potential to be used in large farm animals for production of recombinant human lactoferrin in industrial scale.

Animals↗
PubMed1998

Intraspecies polymorphism of vsp genes and expression profiles of variable surface protein antigens (Vsps) in field isolates of Mycoplasma bovis.

To assess the extent of interstrain variation, 50 isolates of Mycoplasma (M.) bovis including the type strain PG45 were examined for the presence of a family of variable membrane surface lipoproteins (Vsps) and their genes. Southern hybridization using a genomic fragment carrying three distinct vsp genes (vspAEF) revealed a striking heterogeneity, with only 2/50 strains having identical banding patterns. Cluster analysis of the data showed that most isolates from interrelated herds (groups 1, 2 and 3) were combined in a cluster of 50% homology, while isolates from distinct geographical regions (groups 4, 5 and 6) were linked only at 18% homology. Vsp antigen expression was monitored by Western immunoblotting using four specific monoclonal antibodies (MAbs). Resembling the findings at the DNA level, interstrain variation of Vsp expression among groups 1-3 was less pronounced than among non-interrelated isolates from groups 4-6. Ten out of 50 strains did not hybridize with the vspAEF gene probe at high-stringency conditions, 8/50 failed to react with any of the Vsp-related MAbs, and 6/50 proved negative in both assays. Interestingly, most of these isolates produced hybridization signals at low stringency suggesting major distinctions in their vsp gene structure. The extensive evidence obtained on interstrain vsp gene polymorphism and variation in Vsp expression could provide a basis for a future understanding of the pathogenic potential of individual M. bovis strains.

Animals↗
PubMed1995

T-cell receptor alpha and beta chain gene expression in cells infiltrating human cardiac allografts.

Intragraft T-cell receptor (TCR) alpha and beta chain variable region gene expression was analyzed in human cardiac allograft biopsies by reverse transcription polymerase chain reaction. Rearranged TCR alpha and beta chain gene transcripts were detected in all biopsies examined (N = 23), indicating the presence of T cells bearing the alpha/beta TCR even in the absence of microscopically apparent leukocyte infiltration. In this analysis, a broad TCR alpha/beta repertoire in actively rejecting lesions was demonstrated, whereas fewer TCR alpha and beta chain gene families were detected in nonrejecting lesions. The number of expressed TCR V beta chain gene families typically was two- to sixfold higher than that of V alpha chain families in all biopsies tested. This asymmetric relation was present throughout the histologic grading spectrum of the biopsies. Based on these data, the TCR repertoire is heterogenous even in the early stages of mononuclear cell infiltration of the allograft. Also based on the data, the presence of T cells in grafts with minimal cellular infiltrates is not a specific marker of subsequent rejection episode, because T cells were identified in all allograft biopsies.

Aged↗
PubMed2006

[A change in the stability of marker nptII and uidA gene expression in transgenic tobacco plants].

A comparative analysis of variability of heterologous gene expression was conducted in original (T0) transgenic tobacco plants, differing with respect to the copy number of uidA gene (a single and two copies as a direct duplication) within the T-DNA insertion, and among progenies obtained by their self-pollination (T1). Variability of beta-glucuronidase enzyme activity was shown in original transgenic plants or among progenies of the first generation in both groups. It is established that the activity level of beta-glucuronidase among T0 plants with a duplication of the uidA gene is significantly higher (21.9 times) than that determined in transformants with a single-copy gene. In T1 progenies of plant 16.40, carrying a duplication of the uidA gene, a drastic decrease in enzyme activity was observed, which correlated with a decrease or absence of mRNA transcripts, pointing to a disturbance in reporter gene expression. Alterations in the functioning of another heterologous gene (nptII) within the same T-DNA insertion were not observed among progenies of transgenic plant 16.40.

Blotting, Southern↗
PubMed1991

Tandem insertion sequence-like elements define the expression site for variable antigen genes of Borrelia hermsii.

The spirochete Borrelia hermsii avoids the immune response of its mammalian host through multiphasic antigenic variation. Serotype specificity is determined by variable antigens, Vmp proteins, in the outer membrane. Through nonreciprocal recombination between linear plasmids, a formerly silent vmp gene replaces another vmp gene downstream from a common expression site. To further characterize this activating site, we determined the nucleotide sequence of 6.9 kb of the common upstream expression region of strain HS1 of B. hermsii. Preceding the vmp gene promoter and a poly(dT.dA) run were three imperfectly repeated segments of 2 kb. Each of the 2-kb segments contained 1-kb elements with inverted repeats of approximately 0.2 kb each at their termini. The potential of the 1-kb elements to form stem-and-loop structures was demonstrated by heteroduplex analysis. There was no evidence of the presence of the elements elsewhere in the genome of B. hermsii. One or more of these elements may confer the unidirectionality that characterizes vmp gene switches.

Animals↗
PubMed1987

[Inheritance and expression of dominant genes with variable penetration: the evolutionary aspect].

According to up-to-date literature, one of the approaches to elucidating the essence of evolutionary events consists in admitting the importance of events which change genome activity. A crucial way of reorganization of gene activity is the inhetrited activation or inactivation of the genes. "Dormant" gene hypothesis and related data are reviewed in this connection. Most attention is concentrated on the study of inheritance and penetrance of fused gene in mice. By means of individual genetic analysis, it became possible to make a clear-cut distinction between the phenomenon of the lowered gene penetrance and its inherited inactivation. It was shown that low penetrance is a more frequent event which seems to mask the inherited inactivation of the gene. A general scheme of the phenomena studied is proposed. The classical conceptions claiming the existence of a reserve of hereditary variability concern mainly the recessive and codominant mutations. However, the role of dominant and semidominant mutations in the course of functional reconstruction of species may be important. During animal domestication, for example, everyone can see a lot of dominantly inherited characters appearing de novo. D.K. Belyaev's conception about "dormant" gene reserve is of great importance in this connection. Opening of the reserve at a particular evolutionary stage may drastically increase the genetic variability and lead to appearance of evolutionary novelty.

Animals↗
PubMedDate not supplied

Correlation of minimal residual disease by assessing Wilms tumor gene expression and engraftment by variable number of tandem repeats in children with leukemia posthematopoietic stem cell transplantation.

An important measure to ensure successful follow-up in patients with allogeneic stem cell transplant is to evaluate for engraftment. Recent studies have shown that detecting minimal residual disease is important in order to predict early clinical relapse. We followed 88 leukemic patients with pre- and posttransplant Wilms tumor gene (WT1) levels to predict relapse and variable number of tandem repeats (VNTR) for engraftment. We have found that high pretransplant WT1 levels correlated significantly with relapse in all patient groups, but more significantly in the acute nonlymphoblastic leukemia (ANLL) patients. Posttransplant WT1 level correlated with VNTR status such that low WT1 is associated invariably with VNTR of 100% donor origin, while high WT1 is associated with VNTR of 20%. The association is significant in all patients, specifically in ANLL patients. In this preliminary study, we demonstrate that patients harboring detectable levels of WT1 prior to stem cell transplant have a higher chance of relapse, and posttransplant WT1 and VNTR status appeared to be dependent parameters predicting relapse when present in the posttransplant period. By combining 2 highly sensitive molecular techniques, we have found that this combined technique provided us with a promising alternative for overcoming the limitations imposed by each separate procedure. More studies are necessary before we can come to any significant conclusions.

Adolescent↗
PubMed1993

Differential 3' polyadenylation of the Huntington disease gene results in two mRNA species with variable tissue expression.

Recently a novel gene containing a CAG trinucleotide repeat that is expanded on HD chromosomes has been identified(1). This gene was shown to detect a single transcript of 10-11 kb by RNA hybridization. We have however, previously identified three cDNAs which are part of the same gene that have been shown to detect two distinct transcripts of 10 kb and one that is significantly larger(2,3). These different mRNA species could be due to use of alternate transcription start sites, alternate splicing or selection of different polyadenylation sites. We have identified cDNA clones spanning the HD gene including two (HD12 and HD14) that share identical protein coding sequences but differ in size and sequence of their 3' untranslated region. HD14 has 3,360 base pairs of additional sequence distal to the previously published 3' end (1). RNA hybridization has revealed that the larger 13.7 kb fragment is the predominant transcript in human brain. cDNA fragments unique to HD14 detected only the larger transcript. Sequence analysis identified two different putative polyadenylation sequences at position 10,326 and 13,645 of the HD14 cDNA. These findings indicate that the two observed mRNA species originate from a single gene and that differential polyadenylation leads to transcripts of different size. The relative increased abundance of the larger transcript in human brain may provide some insights into the mechanism by which a widely expressed gene may exert tissue specific effects.

Base Sequence↗
PubMed2005

Analysis of the expressed heavy chain variable-region genes of Macaca fascicularis and isolation of monoclonal antibodies specific for the Ebola virus' soluble glycoprotein.

The cynomolgus macaque, Macaca fascicularis, is frequently used in immunological and other biomedical research as a model for man; understanding it's antibody repertoire is, therefore, of fundamental interest. The expressed variable-region gene repertoire of a single M. fascicularis, which was immune to the Ebola virus, was studied. Using 5' rapid amplification of cDNA ends with immunoglobulin (Ig)G-specific primers, we obtained 30 clones encoding full-length variable, diversity, and joining domains. Similar to the human V(H) repertoire, the M. fascicularis repertoire utilized numerous immunoglobulin heavy variable (IGHV) gene fragments, with the V(H)3 (41%), V(H)4 (39%), and V(H)1 (14%) subgroups used more frequently than the V(H)5 (3.9%) or V(H)7 (1.7%) subgroups. Diverse immunoglobulin heavy joining (IGHJ) fragments also appeared to be utilized, including a putative homolog of JH5beta gene segment identified in the related species Macaca mulatta, Rhesus macaque, but not in humans. Although the diverse V region genes in the IgG antibody repertoire of M. fascicularis had likely undergone somatic hypermutations (SHMs), they nevertheless showed high nucleotide identity with the corresponding human germline genes, 80-89% for IGHV and 72-92% for IGHJ. M. fascicularis and human V(H) genes were also similar in other aspects: length of complementarity-determining regions and framework regions, and distribution of consensus sites for SHMs. Finally, we demonstrated that monoclonal antibodies (mAbs) specific for an Ebola protein could be obtained from M. fascicularis tissue samples by phage display technology. In summary, the study provides new insight into the M. fascicularis V region gene repertoire and further supports the idea that macaque-derived mAbs may be of therapeutic value to humans.

Amino Acid Sequence↗
PubMed1996

Expression of the Variable Region Genes of the Monoclonal Antibodies Against Metal-bound Tetrapeptide in E. coli.

The variable region genes of the light and heavy chains obtained from three stems of McAb against metal-bound tetrapeptide were joined into a single chain by a linker. A 39 bp fragment of the N-terminal of CGRP was joined to the C-terminal of the heavy chain to constitute the Lv-linker-Hv-CGRP single chain gene which was cloned into the vector pTC01 and expressed in E. coli 71/18. The molecular weight of the expressed product was approximately 26 kD as shown by SDS-PAGE. Its expression level was about 20%-30% to he total cellular proteins. The product was a soluble protein and showed binding activity with its hapten by indirect ELISA assay.

Journal Article↗
PubMed1986

Preferential expression of variable region heavy chain gene segments by predominant 2,4-dinitrophenyl-specific BALB/c neonatal antibody clonotypes.

The B-cell repertoire in neonatal mice contains predominant clonotypes that are reproducibly expressed at particular times after birth. We have isolated and sequenced heavy and light chain cDNA clones from three 2,4-dinitrophenyl-specific neonatal hybridomas. Two of these hybridomas (TF2-36 and TF5-139) express idiotypes (Ids) that predominate during the first days after birth, and the third hybridoma (TF2-76) expresses an Id that predominates during the second week after birth. The heavy (H) chain variable (V) region of the TF2-76 hybridoma protein is encoded by a member of the 7183 VH family, one of eight families of murine VH genes that have been defined by Brodeur and Riblet [Brodeur, P. H. & Riblet, R. (1984) Eur. J. Immunol. 14, 922-930]. Members of this family have been found to undergo a disproportionately high frequency of rearrangement in fetal and neonatal liver pre-B-cells. Because the 7183 VH family is located close to the H chain joining (J) region gene segments, JH, other workers have proposed that VH rearrangement frequency is related to distance from the JH segments. However, the two earlier-appearing predominant clonotypes expressed by TF2-36 and TF5-139 hybridoma proteins utilize a member of the 36-60 VH family, probably VH 1210.7, which is located distal to the JH gene segments on chromosome 12. Since 20-30% of day 3 dinitrophenyl-specific B cells express either the Id(TF2-36) or the Id(TF5-139), the VH 1210.7 gene must be utilized at high frequency early in development. These results indicate that the utilization of rearranged VH segments is strongly influenced by factors other than distance from JH.

Animals↗
PubMed1994

T cell receptor gene expression and genotypes in celiac disease.

Celiac disease (CD) occurs as a result of an abnormal immune response, within the mucosa of the small bowel, to dietary gliadin peptides. To further characterize the intramucosal lymphocytes in patients with untreated CD, we compared T cell receptor (TCR) variable region gene expression in small bowel biopsies from patients with CD to that of normal small bowel. We also assessed TCR genotypes, using restriction fragment length polymorphisms (RFLPs) spanning the V beta gene locus, comparing 59 CD patients to 64 normals. The abnormal immune response in CD is polyclonal, without evidence of restriction or significantly increased expression of any TCR variable region gene families, compared to normal small bowel. No significant association was found between TCR genotypes, as defined by TCR V beta RFLPs, and CD.

Adult↗
PubMed2005

Transcriptome analysis in primary neural stem cells using a tag cDNA amplification method.

BACKGROUND: Neural stem cells (NSCs) can be isolated from the adult mammalian brain and expanded in culture, in the form of cellular aggregates called neurospheres. Neurospheres provide an in vitro model for studying NSC behaviour and give information on the factors and mechanisms that govern their proliferation and differentiation. They are also a promising source for cell replacement therapies of the central nervous system. Neurospheres are complex structures consisting of several cell types of varying degrees of differentiation. One way of characterising neurospheres is to analyse their gene expression profiles. The value of such studies is however uncertain since they are heterogeneous structures and different populations of neurospheres may vary significantly in their gene expression. RESULTS: To address this issue, we have used cDNA microarrays and a recently reported tag cDNA amplification method to analyse the gene expression profiles of neurospheres originating from separate isolations of the lateral ventricle wall of adult mice and passaged to varying degrees. Separate isolations as well as consecutive passages yield a high variability in gene expression while parallel cultures yield the lowest variability. CONCLUSIONS: We demonstrate a low technical amplification variability using the employed amplification strategy and conclude that neurospheres from the same isolation and passage are sufficiently similar to be used for comparative gene expression analysis.

Animals↗
PubMed2006

Immunoglobulins and immunoglobulin genes of the horse.

Antibodies of the horse were studied intensively by many notable immunologists throughout the past century until the early 1970's. After a large gap of interest in horse immunology, additional basic studies on horse immunoglobulin genes performed during the past 10 years have resulted in new insights into the equine humoral immune system. These include the characterization of the immunoglobulin lambda and kappa light chain genes, the immunoglobulin heavy chain constant (IGHC) gene regions, and initial studies regarding the heavy chain variable genes. Horses express predominately lambda light chains and seem to have a relatively restricted germline repertoire of both lambda and kappa chain variable genes. The IGHC region contains eleven constant heavy chain genes, seven of which are gamma heavy chain genes. It is suggested that all seven genes encoding IgG isotypes are expressed and have distinct functions in equine immune responses.

Animals↗
PubMed1997

Human Fab fragments specific for the Haemophilus influenzae b polysaccharide isolated from a bacteriophage combinatorial library use variable region gene combinations and express an idiotype that mirrors in vivo expression.

To determine whether the human antibody (Ab) repertoire to the Haemophilus influenzae type b capsular polysaccharide (Hib PS) could be studied at the molecular level with phage display technology, we constructed a phage Fab library by using peripheral blood from a vaccinated adult. Phage were selected based on Hib PS binding. Two distinct Hib PS-specific phage clones were identified whose Fab fragments used the same V(H) region paired with two different V(L) regions. The V(L) regions were derived from two independent rearrangements of the A2c gene with Jkappa1, and both contained a nontemplated arginine codon at the V-Jkappa junction. The two A2 V gene segments differed from the A2c germ line sequence in 0 and 5 bases. The V(H) region consisted of the V(H)26 gene segment having 98% identity to the germline nucleotide sequence, a D region of 9 bases, and J(H)4b1. Usage of V(H)26 in combination with A2 V regions containing a junctional arginine is a predominant configuration of naturally occurring Hib PS-specific Abs. Liquid- and solid-phase assays showed that phage-derived Fab reacted with Hib PS and expressed HibId-1, an idiotype associated with the kappaII-A2 V region. These findings extend the database of V region polymorphisms that can contribute to the Hib PS repertoire and demonstrate that Hib PS-specific Fab fragments isolated from combinatorial phage libraries use V gene combinations which mirror the natural repertoire.

Adult↗
PubMed1984

Isolation of hybridomas expressing a specific heavy chain variable region gene segment by using a screening technique that detects mRNA sequences in whole cell lysates.

A technique is described that allows single hybridoma cell colonies to be assayed for the productive rearrangement of a single immunoglobulin variable region (V) gene segment by utilizing expression of V mRNA for analysis. Hybridomas growing in microwell tissue culture plates are lysed in situ, cellular RNA is directly transferred to nitrocellulose by filtration, and specific immunoglobulin mRNA is detected by hybridization of the filter with a DNA probe. The method is simple and sensitive. A single species of mRNA can be detected in a lysate of 1000 cells; 5000 hybridoma colonies can be easily screened per day. The technique has been successfully used to isolate cell lines from nonimmune mice expressing a particular heavy chain variable region (VH) gene segment.

Animals↗