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Activation of the delta-globin gene by the beta-globin gene CACCC motif.

The promoter region of adult beta globin genes in humans and other mammals contains conserved regions of pivotal importance for their regulated tissue specific expression. These include the CACCC and CAAT motifs. The CACCC motif is duplicated in humans and other mammals. The human delta-globin gene lacks these conserved regions and its expression in normal individuals is about 3% that of the beta globin gene. Previous studies have shown that the introduction of the beta-globin CACCC or CAAT can activate the delta-globin gene promoter, but the effect of the distal CACCC element has not yet been tested. In the present study, using site-specific mutagenesis, we have introduced the consensus sequence for the distal and proximal CACCC motif and the CAAT box alone or in combination in the wild-type delta-globin gene promoter. The resulting mutants, as well as the wild type (wt) delta- and beta-globin gene promoters, have been analyzed in a transient expression assay in Cos7, K562, and MEL cell lines. The results show that the CACCC boxes can increase the transcription efficiency of the delta-globin gene promoter in both erythroid and non-erythroid cell systems. The contribution of the two CACCC elements is almost equal in the non-erythroid (Cos7) and erythroid embryonic-fetal cell lines (K562), while the proximal CACCC element is more active in adult erythroid cells (MEL). Nonetheless, duplication of this element does not appear to affect the efficiency of the promoter synergistically. Furthermore, to assess the competitive ability of the delta globin promoter containing the proximal or distal CACCC consensus sequences over the wt beta globin gene promoter, we have carried out transient expression experiments using DNA constructs in which the delta and beta globin gene promoters are linked in cis and are sharing a single enhancer (competitive transient expression). The results show that both CACCC elements are able to activate the delta globin gene promoter in Cos7 and K562 cells, although to a different extent, whereas only the proximal CACCC element is effective in increasing the transcription efficiency in MEL cells. These findings are in agreement with the more severe clinical phenotype produced by the beta-thalassemia mutations affecting the proximal CACCC box as compared with those within the distal CACCC box. The Erythroid Kruppel Like Factor (EKLF) is a nuclear protein restricted to erythroid cells which specifically bind the CACCC box sequence and activate the beta-globin gene. In the present study we carried out transactivation experiments of the mutagenized delta-globin gene promoter by introducing an EKLF expressing construct in erythroid cells. Constructs containing the proximal but not those bearing the distal CACCC element are transactivated. Our results indicate that the proximal CACCC box and, to a lesser extent, also the distal box have a role in the regulated stage specific expression of a beta-like globin gene, and show that the insertion of a single CACCC motif in the delta-globin gene promoter is sufficient to increase its activity. Nevertheless only the delta globin gene promoter containing the proximal CACCC element is able to compete with the wt beta globin gene promoter in the adult erythroid environment. These findings have potential relevance for the future prospective treatment of inherited hemoglobinopathies based on the conversion of the low functioning delta-globin gene into a high functioning beta-like globin gene.

Animals↗
PubMed1995

The organization structure and regulatory elements of Chlamydomonas histone genes reveal features linking plant and animal genes.

The genome of the green alga Chlamydomonas reinhardtii contains approximately 15 gene clusters of the nucleosomal (or core) histone H2A, H2B, H3 and H4 genes and at least one histone H1 gene. Seven non-allelic histone gene loci were isolated from a genomic library, physically mapped, and the nucleotide sequences of three isotypes of each core histone gene species and one linked H1 gene determined. The core histone genes are organized in clusters of H2A-H2B and H3-H4 pairs, in which each gene pair shows outwardly divergent transcription from a short (< 300 bp) intercistronic region. These intercistronic regions contain typically conserved promoter elements, namely a TATA-box and the three motifs TGGCCAG-G(G/C)-CGAG, CGTTGACC and CGGTTG. Different from the genes of higher plants, but like those of animals and the related alga Volvox, the 3' untranslated regions contain no poly A signal, but a palindromic sequence (3' palindrome) essential for mRNA processing is present. One single H1 gene was found in close linkage to a H2A-H2B pair. The H1 upstream region contains the octameric promoter element GGTTGACC (also found upstream of the core histone genes) and two specific sequence motifs that are shared only with the Volvox H1 promoters. This suggests differential transcription of the H1 and the core histone genes. The H1 gene is interrupted by two introns. Unlike Volvox H3 genes, the three sequenced H3 isoforms are intron-free. Primer-directed PCR of genomic DNA demonstrated, however, that at least 8 of the about 15 H3 genes do contain one intron at a conserved position. In synchronized C. reinhardtii cells, H4 mRNA levels (representative of all core histone mRNAs) peak during cell division, suggesting strict replication-dependent gene control. The derived peptide sequences place C. reinhardtii core histones closer to plants than to animals, except that the H2A histones are more animal-like. The peptide sequence of histone H1 is closely related to the V. carteri VH1-II (66% identity). Organization of the core histone gene in pairs, and non-polyadenylation of mRNAs are features shared with animals, whereas peptide sequences and enhancer elements are shared with higher plants, assigning the volvocalean histone genes a position intermediate between animals and plants.

Amino Acid Sequence↗
PubMed1999

Gene conversion of major histocompatibility complex genes is associated with CpG-rich regions.

We examined 32 DNA sequences of mouse and human major histocompatibility complex (MHC) genes believed to have been subjected to gene conversion events. All regions of the mouse H2 genes as well as the human HLA genes which have been implied to be involved in gene conversion events had elevated levels of CpG dinucleotides, whereas the rest of the genes showed extensive CpG suppression. Mouse MHC genes which have been suspected but not directly implied to be involved in gene conversion events also showed elevated levels of CpG dinucleotides. Moreover, both mouse and human MHC genes which have never been suspected of undergoing gene conversion had low levels of CpG throughout the genes. These results indicate that high CpG levels are correlated with gene conversion rather than with polymorphism, as non-polymorphic genes that have been implicated as gene conversion donors also have elevated levels of CpG dimers in the involved regions, whereas polymorphic genes which have never been considered to undergo gene conversion events have a low level of CpG dinucleotides. We also studied the methylation pattern of CpG dimers in the Abk gene by restriction enzyme digestion of mouse testis DNA followed by Southern blot and hybridization to an Abk-specific probe. The examined CpG dimers in prepubescent mice, where the latest germline stages are spermatogonia, leptene, or pachytene, are respectively non-methylated. Accordingly, the CpG dimers appear to be non-methylated in germline DNA from the testis of prepubescent mice, where gene conversions have been reported to occur.

Animals↗
PubMed1997

Evidence for gene conversion in the generation of extensive polymorphism in the promoter of the growth hormone gene.

The human growth hormone gene (GH-N) is located in a cluster of five highly homologous genes that are coordinately expressed in pituitary (GH-N) and in placental tissues (the chorionic-somatomammotropin-like gene, the GH-variant gene and the two chorionic somatomammotropin genes). Sequence analysis from position -162 to position +100 of the GH-N gene has revealed eight nucleotide polymorphisms with no significant difference in frequency between patients affected by isolated growth hormone deficiency and controls. Remarkably, all these variations are located at positions where the GH-N differs from at least one of the other four homologous genes. The analysis of the twelve GH-N haplotypes originating from the combinations of the eight polymorphisms has revealed that not only single variations, but also nucleotide combinations are identical to those of the other placental genes. These findings suggest that whole stretches of the GH-N gene promoter have been replaced by homologous DNA stretches copied from one of the other four loci by repeated gene-conversion-like events, where the GH-N gene has acted as the recipient and the placental genes as donors of the converted sequences. The presence of a Chi-like element also indicates that the GH-N promoter represents a hot spot of gene conversion. Three of these variations cause, in addition, an amino-acid substitution in the GH-gene-derived transcriptional activator gene whose coding sequence overlaps the GH-N promoter. Thus, a DNA region that serves two distintic functions representing the proximal promoter of a gene and the 5' coding region of another gene displays an unusually high degree of polymorphism that has probably arisen because of gene conversion.

Base Sequence↗
PubMed2005

Tendon healing in vitro: modification of tenocytes with exogenous vascular endothelial growth factor gene increases expression of transforming growth factor beta but minimally affects expression of collagen genes.

PURPOSE: It is not clear how the transfer of exogenous growth factor genes to tenocytes affects collagen production. An increase in collagen production enhances the repair but an increase in growth factors that stimulate tissue fibrosis may cause adhesion. Gene therapy is a new way to regulate tendon healing but it has been explored rarely. We genetically modified tenocytes with the vascular endothelial growth factor (VEGF) gene and investigated the expression of the genes for collagen production in an in vitro model of the proliferating tenocytes. METHODS: Tenocytes were obtained from cultures of rat intrasynovial tendons and distributed randomly to 25 dishes. The tenocytes in the experimental dishes (n = 9) were treated for 12 hours with plasmid containing the VEGF complementary deoxyribonucleic acid and then were cultured for 5 days; the tenocytes in the control dishes (n = 8) did not receive the exogenous gene. Tenocytes in the other dishes received exogenous platelet-derived growth factor (PDGF) gene for comparison of the effects of VEGF gene therapy. Efficiency of the gene transfer was evaluated by presence of the transgene in the tenocytes which was detected by reverse transcription polymerase chain reactions. Levels of expression of types I and III collagen and transforming growth factor (TGF)-beta genes were determined by quantitative analysis of the products of reverse transcription polymerase chain reactions. RESULTS: Expression of the TGF-beta gene increased significantly in the cells treated with exogenous VEGF cDNA. Expression of type I and III collagen genes by tenocytes was affected minimally by transfer of the VEGF gene to the tenocytes and was significantly weaker than that stimulated by PDGF gene therapy. Efficient gene transfer was confirmed by the presence of the VEGF complementary deoxyribonucleic acid in the tenocytes receiving the transferred gene. CONCLUSIONS: Transfer of exogenous VEGF gene has very limited effects on the promotion of collagen production in the proliferating tenocytes. This study suggests that VEGF gene therapy is not as beneficial as PDGF gene therapy to tendon healing and may increase the activities of TGF-beta that are associated with adhesion formations.

Animals↗
PubMed2005

Gene expression profile differences in gastric cancer, pericancerous epithelium and normal gastric mucosa by gene chip.

AIM: To study the difference of gene expression in gastric cancer (T), pericancerous epithelium (P) and normal tissue of gastric mucosa (C), and to screen an associated novel gene in early gastric carcinogenesis by oligonucleotide microarray. METHODS: U133A (Affymetrix, Santa Clara, CA) gene chip was used to detect the gene expression profile difference in T, P and C, respectively. Bioinformatics was used to analyze the detected results. RESULTS: When gastric cancer was compared with normal gastric mucosa, 766 genes were found, with a difference of more than four times in expression levels. Of the 766 genes, 530 were up-regulated (Signal Log Ratio (SLR) >2), and 236 were down-regulated (SLR<-2). When pericancerous epithelium was compared with normal gastric mucosa, 64 genes were found, with a difference of more than four times in expression levels. Of the 64 genes, 50 were up-regulated (SLR>2), and 14 were down-regulated (SLR<-2). Compared with normal gastric mucosa, a total of 143 genes with a difference in expression levels (more than four times, either in cancer or in pericancerous epithelium) were found in gastric cancer (T) and pericancerous epithelium (P). Of the 143 genes, 108 were up-regulated (SLR>2), and 35 were down-regulated (SLR<-2). CONCLUSION: To apply a gene chip could find 143 genes associated with the genes of gastric cancer in pericancerous epithelium, although there were no pathological changes in the tissue slices. More interesting, six genes of pericancerous epithelium were up-regulated in comparison with genes of gastric cancer and three genes were down-regulated in comparison with genes of gastric cancer. It is suggested that these genes may be related to the carcinogenesis and development of early gastric cancer.

Epithelium↗
PubMed2006

[The down regulation of VEGF gene expression may link to change the expression profile of genes in leukemia cell line K562].

OBJECTIVE: To explore the potential effects of anti-VEGF hairpin ribozyme gene to gene expression profiles in leukemia cell line K562. METHODS: The lipofectamine mediation was used to transfect the recombinant eukaryotic expression plasmid (pcDNA3-RZ) containing anti-VEGF hairpin ribozyme gene and the non-recombinant vector as control into K562 cells. And the positive clones were screened by G418. Ribozyme gene in K562 cells was confirmed by PCR. Fluorescent real time reverse transcription-PCR(RT-PCR) and Western blotting were employed to detect the expression of VEGF mRNA and protein in leukemia cells. cDNA microarray was used to explore the alteration of gene expression profiles when decreasing VEGF gene expression in leukemia cells. Expression of PCNA and GSN genes were verified by semi-quantitative RT-PCR. RESULTS: The pcDNA3-RZ and pcDNA3 had been transfected into the human leukemia cell line K562 and positive clones been screened by G418. Stable expression of the ribozyme gene in K562 cells was confirmed by PCR. The level of VEGF mRNA and protein decreased dramatically in K562-RZ cells when compared with K562 or K562-PC (K562 cell transfected with empty vector) cells. The gene expression profiles were changed by transfection of anti-VEGF hairpin ribozyme gene into K562 cells. Among 4096 gene clones on the microarray, 191 (4.86%) genes were detected to have the marked changes with 104 down-regulated and 87 up-regulated, that were functionally related to cell cycle progression, gene replication, metabolism, cell apoptosis, cell signal transduction, and oncogenes etc. An increased expression of GSN gene and a decreased expression of PCNA gene in K562/RZ cells have been detected by RT-PCR. CONCLUSION: Down-regulation of VEGF gene by introducing anti-VEGF hairpin ribozyme gene can alter the gene expression profiles in K562 cells, leading to change of cell growth, differentiation and apoptosis in K562/RZ cells.

Down-Regulation↗
PubMed1989

Avian keratin genes. II. Chromosomal arrangement and close linkage of three gene families.

We describe the isolation and characterization of a set of overlapping cosmid clones that contain chicken keratin genes. The 100 kb (1 kb = 10(3) base-pairs) of DNA represented in these clones contains a cluster of 18 feather keratin genes spanning 53 kb of DNA. The feather keratin genes are spaced about 3 kb apart and at least 11 of them have the same transcriptional orientation. Southern analysis using oligonucleotide probes made from highly conserved portions of the 5' non-coding, intron and 3' non-coding regions, respectively, indicate that these sequences have been highly conserved among the gene family as a whole, with only one or two exceptions in each case. The presence of some regularly repeated restriction enzyme sites are indicative of tandem duplication events in the recent history of the feather keratin gene family. The feather keratin gene locus is flanked on both sides by related types of keratin genes. On the 5' side of the feather gene cluster are three keratin genes (designated feather-like) that are located 5 kb from the last feather keratin gene and are spaced about 4 kb apart. On the 3' side of the feather gene cluster, 21 kb from the last feather keratin gene, lies a cluster of four genes that encode claw keratins. These genes are spaced about 1 kb apart and appear to be divergently transcribed. Partial DNA sequence analysis of the feather-like gene lying proximal to the feather keratin gene cluster demonstrated that it encodes a protein of 115 amino acid residues that is 80% homologous to the feather keratins at both the DNA and amino acid sequence levels. The feather-like gene(s) are expressed in both embryonic and adult (post-hatch) chick feathers and at a very low level in embryonic scale tissue. These genes therefore form a new family of feather proteins that is distinct from the previously characterized feather keratins.

Animals↗
PubMed2004

Successful in utero gene transfer using a gene gun in midgestational mouse fetuses.

BACKGROUND/PURPOSE: In utero gene therapy offers a number of potential advantages over postnatal gene therapy. A latest method of gene transfer to fetuses in utero uses a new tool called a gene gun. The gene gun is less invasive and simpler than other in utero methods. The current study was designed to determine whether the gene gun is an effective tool for transferring genes to mouse fetuses in utero. METHODS: Using a gene gun, we transferred plasmids that included enhanced green fluorescent protein (EGFP) genes and cytomegalo virus promoters to the abdominal skin of 40 A/J fetal mice at each of 3 gestational ages (13, 14, or 15 days). Four or 5 days after gene transfer, the number of surviving fetuses was counted, and a color image of EGFP in the skin was analyzed for gene transfer rates by fluorescence microscopy. Survival rates were analyzed using Fisher's Exact test. RESULTS: The mean survival rate was 89.2% (107 of 120) in gene transfer fetuses and 91.7% (55 of 60) in controls. There is no difference in survival rate between gene transfer fetus and control. The highest gene transfer rate was 100% (37 of 37) at the gestational age of 14 days. The rate was 97.1% (34 of 35) at gestational ages of 13 and 15 days. CONCLUSIONS: The results of this study show that in utero gene transfer by gene gun is a less-invasive technique, and the gene gun is an effective tool transferring genes to mouse fetuses in utero.

Animals↗
PubMed1998

Preferential selection of heterologous G3-VP7 gene in the genetic background of simian rotavirus SA11 detected by using a homotypic single-VP7 gene-substitution reassortant.

Introduction of segmented genomes into virion is an important process in viral replication of rotavirus. We previously studied the assortment of the VP7 gene segment (encoding outer capsid protein VP7) in the genetic background of simian rotavirus SA11 (G serotype 3, G3) and found the preferential selection of homologous G3 VP7 gene over VP7 gene of heterologous G serotype (G1, G2 or G4). In the present study, in order to clarify whether or not VP7 gene derived from different G3 rotavirus (heterologous G3-VP7 gene) is also preferentially selected in the SA11 background, a single-VP7 gene-substitution reassortant was prepared from SA11 through multiple steps of coinfection with rotaviruses in vitro. The isolated reassortant, SNR1, possessed VP7 gene derived from canine G3 rotavirus K9 and all other gene segments of SA11 origin, and showed an identical growth characteristic to that of SA11. Amino acid sequence of K9 VP7 gene showed a high degree of identity (93.6%) to SA11 VP7 gene. In analysis by mixed infection and multiple passages of SNR1 and a single VP7 gene (with G1, G2 or G4 specificity) reassortant in the SA11 background, the G3-VP7 gene became predominant at early passage numbers. However, in mixed infection with SA11 and SNR1, homologous G3-VP7 gene (SA11-VP7 gene) was preferentially selected into progenies over heterologous one (K9-VP7 gene). These results together with our previous findings suggested that G3-VP7 gene, irrespective of origin of species, was functionally adapted to the genetic background of SA11, although the homologous gene had a better fit with other SA11 genes than did heterologous one, providing suggestions for efficaciousness of multivalent reassortant rotavirus vaccine.

Amino Acid Substitution↗
PubMed2000

Comparison of the role of dopamine, serotonin, and noradrenaline genes in ADHD, ODD and conduct disorder: multivariate regression analysis of 20 genes.

The present study is based on the proposal that complex disorders resulting from the effects of multiple genes are best investigated by simultaneously examining multiple candidate genes in the same group of subjects. We have examined the effect of 20 genes for dopamine, serotonin, and noradrenergic metabolism on a quantitative score for attention deficit hyperactivity disorder (ADHD) in 336 unrelated Caucasian subjects. The genotypes of each gene were assigned a score from 0 to 2, based on results from the literature or studies in an independent set of subjects (literature-based scoring), or results based on analysis of variance for the sample (optimized gene scoring). Multivariate linear regression analysis with backward elimination was used to determine which genes contributed most to the phenotype for both coding methods. For optimized gene scoring, three dopamine genes contributed to 2.3% of the variance, p = 0.052; three serotonin genes contributed to 3%, p = 0.015; and six adrenergic genes contributed to 6.9%, p = 0.0006. For all genes combined, 12 genes contributed to 11.6% of the variance, p = 0.0001. These results indicate that the adrenergic genes play a greater role in ADHD than either the dopaminergic or serotonergic genes combined. The results using literature-based gene scoring were similar. An examination of two additional comorbid phenotypes, conduct disorder and oppositional defiant disorder (ODD), indicated they shared genes with ADHD. For ODD different genotypes of the same genes were often used. These results support the value of the simultaneous examination of multiple candidate genes.

Adolescent↗
PubMed2006

Uneven size distribution of mammalian genes in the number of tissues expressed and in the number of co-expressed genes.

Tissue specificity, the traditional predictor of gene function, has recently been used to interpret the selective pressure associated with gene architecture. In this work, we examine gene structures and their relation to the number of tissues expressed and to the number of co-expressed genes, using a recent atlas of microarray-based mouse gene expression in 55 normal tissues. We define tissue specificity and expression-pattern specificity according to the number of tissues expressed and the number of co-expressed genes, respectively. We find that, consistent with previous findings, tissue non-specific (housekeeping) genes are short in all gene regions (coding regions, intron, 5' and 3' untranslated regions). However, in contrast to previous suggestion that tissue-specific genes are long, the genes that are the most tissue-specific (expressed only in one tissue) are also short. We further show that both expression-pattern-specific and non-specific genes are long in coding and non-coding regions. The origins for short tissue-specific genes and long expression-pattern-specific genes are not clear. Genes with highly non-specific expression patterns (i.e. genes with a large number of co-expressed genes) are composed of genes that spread all tissues but are overwhelmingly enriched in the central nervous system (e.g. brain). Thus, the large sizes of these genes are possibly related to the functional complexity and/or accelerated evolutions of the central nervous system.

Animals↗
PubMed1993

Using simultaneous, tandem gene replacements to study expression of the multicopy ubiquitin-fusion (FUS) gene family of Trypanosoma cruzi.

Many genes in trypanosomes exist as members of multicopy gene families. Due to this fact it is frequently difficult to determine if specific members of a gene family are expressed. We describe here a strategy for simultaneous tandem gene replacement in T. cruzi which leads to the replacement of the gene of interest by a silent reporter gene, the expression of which can be assayed in stable transformants. To determine if the FUS1 gene (one of 5 copies of the ubiquitin-fusion, FUS, gene family) was expressed, stable G418-resistant transformants were isolated in which the tandemly arrayed CUB2.65 and FUS1 genes were precisely replaced by the neomycin phosphotransferase (neo(r)) and chloramphenicol acetyltransferase (CAT) genes, respectively. All stable clones carrying the tandem gene replacements were shown to express the CAT activity indicating that FUS1 is expressed in mid-log epimastigotes. Northern blot analysis of parasites carrying the tandem gene replacements indicated that at least one other member of the FUS gene family is expressed and that there were no apparent polar effects on the expression of genes downstream of the replacement events. These experiments have demonstrated the utility of tandem gene replacements as a means of inserting a nonselected reporter gene into the chromosome, facilitating the molecular genetic analysis of the expression of multicopy gene families.

Animals↗
PubMed1989

Delineation of regulatory domains of early (beta) and late (gamma 2) genes by construction of chimeric genes expressed in herpes simplex virus 1 genomes.

The expression of the gamma 2 class of viral genes in cells infected with herpes simplex virus 1 requires viral DNA synthesis and functional viral products made earlier in infection. To identify the sequences required for gamma 2 gene expression, we constructed recombinant viruses in which regions of the thymidine kinase gene (tk), a beta gene normally expressed early in infection, were replaced by specific domains of a gamma 2 gene. The phenotypic attributes examined were (i) sensitivity or resistance of expression in cells exposed to sufficient phosphonoacetate to block viral DNA synthesis, properties of gamma 2 and beta genes, respectively; (ii) expression early in infection, a property of beta genes; and (iii) expression late in infection, a property of gamma 2 genes. We report that replacement of nucleotides -200 to +51 of tk with nucleotides -77 to +104 of the gamma 2 gene conferred upon tk all of the tested attributes of gamma 2 genes. The tk sequence in the 5' transcribed noncoding domain downstream of nucleotide +51 played no apparent role in the expression of the chimeric genes. Similarly, tk sequence downstream of -16 and gamma 2 sequence upstream of -12, when juxtaposed in correct orientations, yielded a chimeric gene that was poorly expressed. In contrast, the chimeric gene consisting of tk sequence upstream of -16 fused to gamma 2 sequence downstream of -12 had the attributes of both beta and gamma 2 genes in that it was expressed both early and late in infection and was partially resistant to phosphonoacetate. The capacity for expression late in infection encoded in the gamma 2 5' transcribed noncoding domain was observed in cells infected with a recombinant virus in which gamma 2 nucleotides +17 to +104 were inserted into the 5' transcribed noncoding domain of the tk gene. We conclude that whereas in the beta genes exemplified by the tk gene the regulatory domains are mainly upstream from nucleotide +51, the sequence(s) that confers gamma 2 regulation is downstream from the TATAA box.

Animals↗
PubMed1988

Structure of the gene for the transition state regulator, abrB: regulator synthesis is controlled by the spo0A sporulation gene in Bacillus subtilis.

Sporulation begins coincidentally with the expression of several stationary-phase-associated gene products during the transition state of a culture from exponential to stationary phase. Mutations in the stage 0 sporulation genes prevent the expression of these gene products in addition to blocking sporulation. Suppressor mutations in the abrB gene, in a spo0 background, restore stationary-phase-associated gene expression but not sporulation. The nature of the abrB gene product was investigated by isolating and sequencing the abrB gene. The abrB gene coded for a 96-amino-acid protein (molecular weight 10773) and contained a helix-turn-helix structure common to DNA binding proteins. Analysis of expression of the abrB gene using lacZ transcription fusions and direct measurement of mRNA content by hybridization showed that the spo0A gene repressed transcription of the abrB gene. Primer extension analysis of abrB gene mRNA revealed two initiation sites. The downstream site was dramatically repressed in spo0A+ strains, while the upstream site appeared not to be regulated by spo0A. Five abrB mutant alleles were cloned and sequenced. One mutation, abrB4, resided within the structural gene and continued to overexpress abrB messenger RNA from both promoters. A promoter mutation, abrB15, reduced transcription from the downstream promoter but not the upstream promoter. Thus, the phenotype of abrB mutations results from inactivation of the abrB gene product or by prevention of its overexpression. The results suggest that the abrB gene codes for a regulator which controls several genes whose products are normally produced during the transition phase between active growth and sporulation. The level of this regulator is, in turn, controlled by the spo0A gene. The pleiotropic phenotypes of spo0A mutants result from uncontrolled overexpression of the abrB regulator.

Amino Acid Sequence↗
PubMed1999

Induction of endogenous genes following infection of human endothelial cells with an E1(-) E4(+) adenovirus gene transfer vector.

Recombinant adenovirus (Ad) gene transfer vectors are effective at transferring exogenous genes to a variety of cells and tissue types both in vitro and in vivo. However, in the process of gene transfer, the Ad vectors induce the expression of target cell genes, some of which may modify the function of the target cell and/or alter the local milieu. To develop a broader understanding of Ad vector-mediated induction of endogenous gene expression, genes induced by first-generation E1(-) E4(+) Ad vectors in primary human umbilical vein endothelial cells were identified by cDNA subtraction cloning. The identified cDNAs included signaling molecules (lymphoid blast crisis [LBC], guanine nucleotide binding protein alpha type S [Galpha-S], and mitogen kinase [MEK5]), calcium-regulated/cytoskeletal proteins (calpactin p11 and p36 subunits, vinculin, and spinocerebellar ataxia [SCA1]), growth factors (insulin-like growth factor binding protein 4 and transforming growth factor beta2), glyceraldehyde-6-phosphate dehydrogenase, an expressed sequence tag, and a novel cDNA showing homology to a LIM domain sequence. Two- to sevenfold induction of the endogenous gene expression was observed at 24 h postinfection, and induction continued up to 72 h, although the timing of gene expression varied among the identified genes. In contrast to that observed in endothelial cells, the Ad vector-mediated induction of gene expression was not found following Ad vector infection of primary human dermal fibroblasts or human alveolar macrophages. Empty Ad capsids did not induce endogenous gene expression in endothelial cells. Interestingly, additional deletion of the E4 gene obviated the upregulation of genes in endothelial cells by the E1(-) E3(-) Ad vector, suggesting that genes carried by the E4 region play a central role in modifying target cell gene expression. These findings are consistent with the notion that efficient transfer of exogenous genes to endothelial cells by first-generation Ad vectors comes with the price that these vectors also induce the expression of a variety of cellular genes.

Adenovirus E1 Proteins↗
PubMed2006

Conditional gene expression in the mouse using a Sleeping Beauty gene-trap transposon.

BACKGROUND: Insertional mutagenesis techniques with transposable elements have been popular among geneticists studying model organisms from E. coli to Drosophila and, more recently, the mouse. One such element is the Sleeping Beauty (SB) transposon that has been shown in several studies to be an effective insertional mutagen in the mouse germline. SB transposon vector studies have employed different functional elements and reporter molecules to disrupt and report the expression of endogenous mouse genes. We sought to generate a transposon system that would be capable of reporting the expression pattern of a mouse gene while allowing for conditional expression of a gene of interest in a tissue- or temporal-specific pattern. RESULTS: Here we report the systematic development and testing of a transposon-based gene-trap system incorporating the doxycycline-repressible Tet-Off (tTA) system that is capable of activating the expression of genes under control of a Tet response element (TRE) promoter. We demonstrate that the gene trap system is fully functional in vitro by introducing the "gene-trap tTA" vector into human cells by transposition and identifying clones that activate expression of a TRE-luciferase transgene in a doxycycline-dependent manner. In transgenic mice, we mobilize gene-trap tTA vectors, discover parameters that can affect germline mobilization rates, and identify candidate gene insertions to demonstrate the in vivo functionality of the vector system. We further demonstrate that the gene-trap can act as a reporter of endogenous gene expression and it can be coupled with bioluminescent imaging to identify genes with tissue-specific expression patterns. CONCLUSION: Akin to the GAL4/UAS system used in the fly, we have made progress developing a tool for mutating and revealing the expression of mouse genes by generating the tTA transactivator in the presence of a secondary TRE-regulated reporter molecule. A vector like the gene-trap tTA could provide a means for both annotating mouse genes and creating a resource of mice that express a regulable transcription factor in temporally- and tissue-specific patterns for conditional gene expression studies. These mice would be a valuable resource to the mouse genetics community for purpose of dissecting mammalian gene function.

Animals↗
PubMed2003

Organ-specific gene expression in the rhesus monkey eye following intravenous non-viral gene transfer.

PURPOSE: The transfer of exogenous genes to the entire retina and other ocular structures is possible with a vascular route of gene delivery using a non-viral gene transfer method. The present studies examine the extent to which either beta-galactosidase or luciferase expression plasmids are targeted to the retina in the adult rhesus monkey following intravenous administration. In addition, these studies examine the pattern of organ expression of the transgene in the rhesus monkey depending on whether the plasmid is under the influence of a widely expressed promoter, the SV40 promoter, or an ocular-specific promoter, the opsin promoter. METHODS: The plasmid DNA with either the SV40 or opsin promoter is encapsulated in the interior of 85 nm pegylated immunoliposomes (PILs), which are targeted across the blood-retinal barrier and into ocular cells with a monoclonal antibody to the human insulin receptor. Following a single intravenous injection of the PIL carrying the transgene, the animals were sacrificed 2, 7, or 14 days later for the measurement of beta-galactosidase or luciferase gene expression in the monkey eye and peripheral organs. RESULTS: Histochemistry showed expression of the beta-galactosidase gene throughout the entire primate retina including the photoreceptor cells with either an SV40 or a bovine opsin promoter. Whereas the SV40 promoter enables gene expression in other organs of the primate (brain, liver, spleen), the opsin promoter restricted trans-gene expression to the primate eye, as there was no gene expressed in other organs. The retinal luciferase activity at 2 days after administration was 9.6+/-0.4 pg luciferase/mg protein, and at 14 days after administration was still comparable to maximal levels of luciferase gene expression in the mouse or rat. Confocal microscopy with antibodies to the insulin receptor and to beta-galactosidase demonstrated co-localization in the retina, with high expression of the trans-gene and the insulin receptor in the inner segments of the photoreceptor cells. CONCLUSIONS: The PIL non-viral gene transfer technology makes possible adult transgenics in 24 h. Ectopic expression of exogenous genes in organs other than the target organ is made possible with the use of organ specific promoters, and gene expression in the primate is restricted to the eye when the trans-gene is under the influence of the opsin promoter. Plasmid-based gene expression is still in the therapeutic range for 2-3 weeks after a single intravenous administration. Exogenous genes are expressed throughout the entire primate retina following the delivery of the gene to the eye via a trans-vascular route.

Animals↗