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K M Klucher

Publications and source records attributed to K M Klucher.

12 recordsLinked to original sources
PubMed2001

Autocrine and paracrine effects of an ES-cell derived, BCR/ABL-transformed hematopoietic cell line that induces leukemia in mice.

During differentiation in vitro, Embryonic Stem (ES) cells generate both primitive erythroid and definitive myeloid lineages in a process that mimics hematopoiesis in the mammalian yolk sac. To investigate leukemic transformation of these embryonic hematopoietic progenitors, we infected differentiating cultures of ES cells with the Chronic Myeloid Leukemia-specific BCR/ABL oncoprotein. Following a period of liquid culture, we isolated two transformed subclones, EB57 and EB67, that retained characteristics of embryonic hematopoietic progenitors and induced a fatal leukemia in mice characterized by massive splenomegaly and granulocytosis. Histopathology of the spleen revealed an abundance of undifferentiated blast-like cells. Investigation of the clonal origins of the granulocytes in the peripheral blood demonstrated that the injected donor cells contributed modestly to the granulocyte population while the majority were host-derived. EB57 secretes IL-3 and unidentified cytokines that can stimulate autocrine and paracrine cell proliferation, presumably accounting for the reactive granulocytosis in diseased mice. These BCR/ABL transformed hematopoietic derivatives of ES cells recapitulate the relationship of BCR/ABL expression to IL-3 production that has been described for primitive hematopoietic progenitors from human CML patients, and illustrates the potential for autocrine and paracrine effects of BCR/ABL-infected cells in murine models.

Animals↗
PubMed1999

The BCR/ABL oncogene alters the chemotactic response to stromal-derived factor-1alpha.

The chemokine stromal-derived factor-1alpha (SDF-1alpha) is a chemoattractant for CD34(+) progenitor cells, in vitro and in vivo. The receptor for SDF-1alpha, CXCR-4, is a 7 transmembrane domain receptor, which is also a coreceptor for human immunodeficiency virus (HIV). Here we show that transformation of hematopoietic cell lines by BCR/ABL significantly impairs their response to SDF-1alpha. Three different hematopoietic cell lines, Ba/F3, 32Dcl3, and Mo7e, were found to express CXCR-4 and to respond to SDF-1alpha with increased migration in a transwell assay. In contrast, after transformation by the BCR/ABL oncogene, the chemotactic response to SDF-1alpha was reduced in all 3 lines. This effect was directly due to BCR/ABL, because Ba/F3 cells, in which the expression of BCR/ABL could be regulated by a tetracycline-inducible promoter, also had reduced chemotaxis to SDF-1alpha when BCR/ABL was induced. The reduced response to SDF-1alpha was not due to an inability of BCR/ABL-transformed cell lines to migrate in general, as spontaneous motility of BCR/ABL-transformed cells was increased. In mice, injection of SDF-1alpha into the spleen resulted in a transient accumulation of untransformed Ba/F3 cells, but not Ba/F3. p210(BCR/ABL) cells administered simultaneously. The mechanism may involve inhibition of CXCR-4 receptor function, because in BCR/ABL-transformed cells, CXCR-4 receptors were expressed on the cell surface, but SDF-1alpha calcium flux was inhibited. Because SDF-1alpha and CXCR-4 are felt to be involved in progenitor cell homing to marrow, the abnormality decribed here could contribute to the homing and retention defects typical of immature myeloid cells in chronic myelogenous leukemia.

Animals↗
PubMed1998

Secondary mutation maintains the transformed state in BaF3 cells with inducible BCR/ABL expression.

The BCR/ABL gene product of the Philadelphia (Ph) chromosome induces chronic myelogenous leukemia (CML). We generated a hematopoietic cell line, TonB210.1, with tetracycline-dependent BCR/ABL expression to investigate the pathways by which BCR/ABL transforms cells. TonB210.1 demonstrates conditional growth factor independence in tissue culture and rapidly forms tumors in mice fed the tetracycline analog doxycycline. The tumors regress completely upon doxycycline withdrawal, but ultimately reform in all animals. After a long latency, tumors also develop in animals never exposed to doxycycline. Subclones of TonB210.1 established from doxycycline-independent tumors demonstrate distinct mechanisms of transformation. Most subclones manifest increased basal levels of BCR/ABL expression; some have lost the capacity to augment expression upon induction, whereas others remain inducible. More interestingly, some subclones maintain tight conditional expression of BCR/ABL and are therefore transformed by secondary mechanisms that no longer require BCR/ABL expression. These subclones show constitutive phosphorylation of the STAT5 protein, suggesting that activating mutations have occurred upstream in the signaling pathway to STAT5. The tight conditional expression of BCR/ABL in the TonB210.1 cell line affords the opportunity to study several interesting aspects of the biology of BCR/ABL, including activation of critical signaling pathways and transcriptional programs, and its potential role in genomic instability.

Animals↗
PubMed1997

A novel method to isolate cells with conditional gene expression using fluorescence activated cell sorting.

An inducible expression system using control elements of the tetracycline resistance operon has recently shown promise for conditional gene expression of any gene of interest. However, intensive screening of multiple independent clones is often required to find cell lines with optimal induction characteristics. By coupling expression of the gene of interest with a fluorescent marker, we have developed a novel fluorescence activated cell sorting (FACS) based strategy to isolate cells with desirable expression characteristics, thus alleviating the laborious isolation and analysis of multiple independent clones.

Animals↗
PubMed1996

The AINTEGUMENTA gene of Arabidopsis required for ovule and female gametophyte development is related to the floral homeotic gene APETALA2.

Ovules play a central role in plant reproduction, generating the female gametophyte within sporophytic integuments. When fertilized, the integuments differentiate into the seed coat and support the development of the embryo and endosperm. Mutations in the AINTEGUMENTA (ANT) locus of Arabidopsis have a profound effect on ovule development. Strong ant mutants have ovules that fail to form integuments or a female gametophyte. Flower development is also altered, with a random reduction of organs in the outer three whorls. In addition, organs present in the outer three floral whorls often have abnormal morphology. Ovules from a weak ant mutant contain both inner and outer integuments but generally fail to produce a functional female gametophyte. We isolated the ANT gene by using a mutation derived by T-DNA insertional mutagenesis. ANT is a member of a gene family that includes the floral homeotic gene APETALA2 (AP2). Like AP2, ANT contains two AP2 domains homologous with the DNA binding domain of ethylene response element binding proteins. ANT is expressed most highly in developing flowers but is also expressed in vegetative tissue. Taken together, these results suggest that ANT is a transcription factor that plays a critical role in regulating ovule and female gametophyte development.

Amino Acid Sequence↗
PubMed1993

In vivo and in vitro analysis of transcriptional activation mediated by the human cytomegalovirus major immediate-early proteins.

To define mechanistically how the human cytomegalovirus (HCMV) major immediate-early (IE) proteins induce early-gene transcription, the IE1 72-kDa protein, the IE2 55-kDa protein, and the IE2 86-kDa protein were analyzed for their ability to activate transcription from an HCMV early promoter in vivo and in vitro. In transient-expression assays in U373MG astrocytoma/glioblastoma and HeLa cells, only the IE2 86-kDa protein was able to activate the HCMV early promoter to high levels. In HeLa cells, the IE1 72-kDa protein was able to activate the promoter to a low but detectable level, and the level of promoter activity observed in response to the IE2 86-kDa protein was increased synergistically following cotransfection of the constructs expressing both IE proteins. To examine the interaction of the HCMV IE proteins with the RNA polymerase II transcription machinery, we assayed the ability of Escherichia coli-synthesized proteins to activate the HCMV early promoter in nuclear extracts prepared from U373MG cells, HeLa cells, and Drosophila embryos. The results of the in vitro experiments correlated well with those obtained in vivo. The basal activity of the promoter was minimal in both the HeLa and U373MG extracts but was stimulated 6- to 10-fold by the IE2 86-kDa protein. With a histone H1-deficient extract from Drosophila embryos, the HCMV early promoter was quite active and was stimulated two- to fourfold by the IE2 86-kDa protein. Addition of histone H1 at 1 molecule per 40 to 50 bp of DNA template significantly repressed basal transcription from this promoter. However, the IE2 86-kDa protein, but none of the other IE proteins, was able to counteract the H1-mediated repression and stimulate transcription at least 10- to 20-fold. The promoter specificity of the activation was demonstrated by the inability of the IE2 86-kDa protein to activate the Drosophila Krüppel promoter in either the presence or absence of histone H1. These results suggest that one mechanism of transcription activation by the IE2 86-kDa protein involves antirepression.

Base Sequence↗
PubMed1992

An AP-1 binding site is the predominant cis-acting regulatory element in the 1.2-kilobase early RNA promoter of human cytomegalovirus.

To extend our analysis of the regulation of human cytomegalovirus (HCMV) early gene expression, we examined a transcription unit located in the terminal repeats of the long segment of the viral genome. This region encodes a major 1.2-kb RNA which is induced at early times in infection but undergoes its largest increase in abundance after the onset of viral DNA replication. To identify the important cis-acting regulatory elements for this gene, two constructs were prepared for use in transient expression assays. One contained 413 bp of the upstream sequence and 43 bp of the leader sequence fused to the gene for chloramphenicol acetyltransferase (CAT). The second construct included 1,722 bp upstream of the start site of the 1.2-kb RNA, the entire transcribed region with an additional 166-bp insert derived from the CAT gene as an assayable marker, and 2,393 bp downstream of the polyadenylation signal. Both constructs were individually transfected into human fibroblast cells, and the cells were infected with HCMV. RNA specified by the hybrid construct was initiated at the correct position and accumulated with the same kinetics as the authentic viral transcript at early times in the infection but did not undergo the increase in abundance at late at late times. By 5'-end-deletion analysis, we determined that the promoter for the 1.2-kb RNA contains a number of cis-acting elements, the most significant of which are the TATA-like sequence CATAAA at -30 and a sequence corresponding to the binding site for the transcription factor AP-1 at -75. Using extracts prepared from HeLa cells as well as from infected and uninfected fibroblasts in gel retardation assays, we obtained evidence for the specific interaction of a cellular factor(s) with the AP-1 binding site. The pattern of binding differed in the HeLa and fibroblast cells but did not change as a function of the HCMV infection. However, the functional importance of the AP-1 binding site and its key role in the regulation of the 1.2-kb RNA was supported by analysis of constructs containing specific point mutations at this site in gel retardation and transient expression assays. Site-specific mutations in the AP-1 consensus sequence, which resulted in the complete loss of binding to cellular factors, eliminated the basal activity and reduced the inducible promoter activity by eightfold.

Antigens, Viral↗
PubMed1990

The human cytomegalovirus 2.7-kilobase RNA promoter contains a functional binding site for the adenovirus major late transcription factor.

We have examined the factors which influence the expression of a major 2.7-kilobase (kb) early transcript encoded by the long repeat of the human cytomegalovirus (HCMV) strain AD169 genome. Previously, by deletion analysis, we determined that the promoter for this early RNA consisted of multiple cis-acting elements (Klucher et al., J. Virol. 63:5334-5343, 1989). Using extracts prepared from HeLa cells as well as from infected and uninfected foreskin fibroblasts, we also obtained evidence for the interaction of a cellular factor with one of these elements. In this study, we have further defined the specificity and functional importance of this binding. On the basis of DNase I footprinting and methylation interference assays, we localized the site of interaction to a region (nucleotides -113 to -106 relative to the mRNA start site) which contains homology to the binding site for the adenovirus major late transcription factor (MLTF), also referred to as the upstream stimulatory factor (USF). The contact points of binding between the cellular factor and the guanine residues within this segment were consistent with the pattern of binding for USF/MLTF. Additionally, by using oligonucleotides containing the binding sites for USF/MLTF from the adenovirus major late promoter and the HCMV 2.7-kb RNA promoter as competitors in gel retardation assays, we were able to show that USF/MLTF bound to the two promoters with similar affinity. Correlation of the binding activity with in vivo functional importance was provided by mutagenesis and transient-expression assays. A point mutation within the HCMV USF/MLTF site lowered the affinity of binding 5- to 10-fold and decreased the inducible activity of the HCMV 2.7-kb RNA promoter by approximately 50%. Furthermore, the addition of the HCMV USF/MLTF site to a minimal 2.7-kb RNA promoter containing only the TATA sequence resulted in an increase in HCMV inducible transcriptional activity of 6- to 20-fold. However, the HCMV USF/MLTF site could not functionally substitute for the TATA sequence. These studies further support the idea that for maximal response to the HCMV infection, the 2.7-kb RNA promoter requires multiple cis-acting sequences, two of which include the binding sites for USF/MLTF and TFIID.

Adenoviridae↗
PubMed1989

Sequences in the human cytomegalovirus 2.7-kilobase RNA promoter which mediate its regulation as an early gene.

We have studied the regulation of expression of a major human cytomegalovirus (HCMV) early transcription unit located within the long repeat of the strain AD169 genome. This region specified a 2.7-kilobase RNA which underwent its largest increase in abundance between 8 and 14 h postinfection. To study the regulation of this gene, its promoter was cloned 5' of the gene for chloramphenicol acetyltransferase (CAT) for use in transient expression assays. A construct containing 651 base pairs of upstream sequence and 54 base pairs of leader sequence was transfected into human fibroblast cells, followed by HCMV infection. Analysis of the steady-state levels of RNA expressed from this hybrid gene indicated that it accumulated with the same kinetics as the authentic viral transcript early in the infection. Cotransfection of human fibroblasts with the 2.7-kilobase RNA promoter-CAT construct and plasmids containing different HCMV immediate-early (IE) genes showed that the region of the HCMV genome encoding the transcription units corresponding to IE1 and 2 and the 5' end of IE3 is capable of stimulating promoter activity but not to the same extent as HCMV infection. To define important cis-acting regulatory elements in the promoter, a series of 5' deletion mutants was constructed. Transient expression analysis showed a stepwise reduction in inducible CAT activity, suggesting the presence of multiple regulatory sites. To further characterize the nature of these sites, we used gel mobility shift assays to study DNA-protein interactions occurring within this promoter sequence. With nuclear extracts prepared from HeLa cells as well as from infected and uninfected human foreskin fibroblasts, we found specific binding of a cellular factor to a region of the promoter important in HCMV inducible activity. This region contains a palindromic octamer with homology to the binding site of the cellular factor USF/MLTF.

Base Sequence↗
PubMedDate not supplied

A gene encoding a host-specific elicitor protein of Phytophthora parasitica.

Extracellular elicitor proteins (elicitins) from Phytophthora species induce local and distal defense responses specifically in plants of the Solanaceae and Cruciferae. Based on elicitin amino acid sequences, elicitin-coding sequences from P. parasitica were amplified by the polymerase chain reaction. A genomic clone containing a complete elicitin gene, parA1, was isolated and sequenced. Elicitin was confirmed to be encoded as a precursor protein containing a 20-amino acid signal peptide that is processed before secretion. Bacterial expression of the cloned elicitin gene as a translational fusion protein containing glutathione S-transferase yielded a biologically active protein capable of inducing a hypersensitive response in tobacco, suggesting that fungus-specific postranslational modifications of elicitin are not required for its activity. Southern blot analysis indicated that elicitin genes occur as a multigene family (at least two to 10 copies) in P. parasitica, P. capsici, P. citricola, P. citrophthora, P. cryptogea, P. drechsleri, P. megasperma, and P. palmivora. Some isolates of P. parasitica that did not produce elicitins still contained elicitin-coding sequences but did not accumulate elicitin mRNA.

Algal Proteins↗