Targeted deletion of a GA tract by S1 nuclease.
Explore the source record for details and available documents.
Base Sequence↗
Biomedical subjects
B Barbeau
Publications and source records attributed to B Barbeau.
Enhancement of HIV-1-induced syncytium formation in T cells by the tyrosyl kinase p56lck.
The CD4 glycoprotein is the primary cellular receptor for human immunodeficiency virus type 1 (HIV-1) and has also been reported to be physically associated with p56lck, a tyrosyl protein kinase p56lck is a member of the src family of nonreceptor protein-tyrosine kinases and is expressed predominantly in T lymphocytes. Our objective was to study the effect of p56lck on the biology of HIV-1. For this purpose, we have stably transfected two human p56lck negative T cell lines (C8166-45 and MT-2) with plasmids encoding for this cellular protein. Following coculture with HIV-1-infected cells or infection with cell-free virus, p56lck-expressing cell lines showed a greater propensity for virus-mediated syncytium formation than parental p56lck-negative cells. The enhancement of HIV-1-induced syncytium formation was not associated with the kinase activity of p56lck, as demonstrated by experiments using a kinase-deficient mutant. However, the physical interaction between CD4 and p56lck was shown to be necessary to obtain the enhancement of syncytium formation since a mutated version of p56lck, which is deficient in its capacity to associate with CD4, did not lead to an increase in virus-mediated cell-to-cell fusion events. Finally, we determined that cells transfected with wild-type and kinase-negative mutant p56lck showed a reduced rate of CD4 endocytosis compared to parental p56lck-negative cells. Together, these results suggest that p56lck can be seen as an accessory molecule facilitating HIV-1-mediated syncytium formation in T cells by a mechanism involving the stabilization of the CD4 molecule at the cell surface.
Cell Line↗
Binding of HIV-1 to its receptor induces tyrosine phosphorylation of several CD4-associated proteins, including the phosphatidylinositol 3-kinase.
Cell surface CD4 molecules are known to be important in several physiological responses of T lymphocytes. The use of human immunodeficiency virus (HIV) particles or purified gp120 molecules as CD4 cross-linking agents has been shown to result in a cascade of intracellular biochemical events. In addition, we and other have provided evidence suggesting that virus-mediated CD4 multimerization can lead to modulation of HIV-1 long terminal repeat-dependent activity and virus production. We were thus interested in measuring the effect of HIV-1 particles on intracellular tyrosine-phosphorylation levels, mostly of CD4-associated proteins. Using the T cell line CEM-T4, we observed that HIV-1 induces an increase in tyrosine phosphorylation of four major proteins physically complexed to the CD4 molecule. Immunoblot analysis permitted the identification of two of these proteins, p56lck and phosphatidylinositol 3-kinase (PI 3-kinase) p85 alpha. No concomitant variation in the level of these two CD4-associated proteins was observed after HIV-1-induced CD4 cross-linking. To our knowledge, this is the first report linking HIV-1-mediated CD4 multimerization to an increase in tyrosine phosphorylation of the PI 3-kinase complex. The four CD4-associated molecules described in this report are most likely implicated in virus-induced CD4-linked signaling events.
Animals↗
Triplex-forming oligonucleotides with unexpected affinity for a nontargeted GA repeat sequence.
We examined the affinity and the specificity of triplex formation for different purine ODNs directed against two portions of a purine sequence derived from the mouse fli-1 gene. As expected, the ODNs antiparallel to the purine strand of their target can form triplex DNA. One parallel ODN showed binding to its target sequence. We explain this unusual binding by an interaction of the ODN with a GA repetition present in the sequence. We further examined the interaction of this ODN with a target composed of 14 GA repetitions. Unexpectedly, one ODN shows higher affinity for a partially complementary GA target relative to its completely complementary target. For another ODN, the binding to the GA target is weaker and might involve skipping of bases in a way that resembles alternate strand triplex formation.
Animals↗
Characterization of the human and mouse Fli-1 promoter regions.
Overexpression of the Fli-1 gene has been shown to be involved in retrovirus-induced mouse tumors. Cloning of the 5' flanking sequence of the mouse and human Fli-1 exon 1 was performed. At least two major transcription initiation sites were localized respectively at 143 and 114 nucleotides upstream of the previously defined mouse Fli-1 cDNA 5' end. The sequences flanking the CAP sites show good conservation between human and mouse (94%). The promoter region contains a potential TATA box lying 30 bp from one of the major identified CAP sites. Several conserved elements, such as GATA, EBS, GC rich, AP-2, AP-3 elements and a repetition of GA were observed next to the two major CAP sites. Furthermore, this latter was shown to form a H-DNA structure in vitro by S1 nuclease sensitivity experiments. The highly conserved 5' non-translated region of exon 1 is predicted to form a very stable hairpin structure which could regulate the Fli-1 expression at the post-transcriptional level. In Cas-Br-E-induced tumors, all the proviruses are found clustered within 35 nucleotides directly upstream the Fli-1 ATG start codon, thus deleting the hairpin structure from the transcript. Promoter activity was tested using the CAT reporter gene transfected in mouse and human erythroid cell lines. No promoter activity could be detected with various mouse Fli-1 promoter-CAT constructs containing 600 bp of the 5' flanking region, the complete exon 1, the 5' end of intron 1 and/or retroviral LTR sequence. Constructions of the human homologue containing nearly 1.5 kbp of Fli-1 5' flanking region was also inactive in transfected cells. These results suggest that multiple levels of regulation might control the Fli-1 expression.
Animals↗
Repression of human immunodeficiency virus type 1 long terminal repeat-driven gene expression by binding of the virus to its primary cellular receptor, the CD4 molecule.
We have previously postulated that the binding of the human immunodeficiency virus type 1 (HIV-1) to cell surface CD4 induces signal transduction pathways that down-modulate production of progeny virions in acutely infected T cells (M. Tremblay, S. Meloche, S. Gratton, M. A. Wainberg, and R.-P. Sékaly, EMBO J. 13:774-783, 1994). To evaluate the possibility that CD4 cross-linking might indeed affect viral gene expression, we have introduced a molecular construct made of the luciferase reporter gene placed under the control of the regulatory elements of HIV-1 in several CD4-positive T-cell lines. We found that cross-linking of CD4 with defective HIV-1 particles and heat-inactivated viruses inhibits long terminal repeat-dependent luciferase expression. Experiments revealed that the gp120-CD4 interaction was necessary to repress HIV-1 long terminal repeat-dependent luciferase activity. The cytoplasmic domain of CD4 was also found to be required for this effect to occur. The virus-mediated signal transduction was shown to be mediated via p56lck-dependent and -independent pathways. These results indicate that the earliest event in the HIV-1 replicative cycle, namely, the binding of the virus to its cellular receptor, can lead to signal transduction culminating in down-modulation of viral gene expression. Thus we propose that defective viruses could regulate the pathogenesis of HIV disease as they constitute the vast majority of circulating HIV-1 particles.
CD4 Antigens↗
Experimental bias in the evaluation of the cellular transient expression in DNA co-transfection experiments.
beta-Galactosidase (beta-gal) expressing vectors are commonly used to standardize the transfection efficiency in transient expression experiments. In the Chinese hamster ovary (CHO) cell line, we transfected beta-gal expressing vectors in combination with different plasmid DNAs. We reported here that the presence of specific DNAs led to statistically significant variations in the beta-gal expression level. Therefore, the measure of beta-gal activity is not necessarily an accurate method to monitor transfection efficiency, and its use to normalize the expression from reporter genes could be questionable.
Animals↗
Expression and DNA rearrangement of proto-oncogenes in Cas-Br-E-induced non-T-, non-B-cell leukemias.
The Cas-Br-E murine leukemia virus is a non-defective retrovirus that induces non-T-, non-B-cell leukemias in susceptible NIH/Swiss mice. A collection of tumors was examined for genomic DNA structure and RNA expression of known or putative proto-oncogenes and one tumor-suppressor gene, with the aim of identifying genes involved in Cas-Br-E-induced non-T-, non-B-cell leukemogenesis. Fli-1, p53, and Evi-1 were found to be rearranged in 72%, 23%, and 18% of the tumors, respectively, whereas no DNA alteration were detected for c-myc, c-myb, Pim-1, Evi-2, and EpoR genes. Evi-1 rearrangements are rarely associated with p53 or Fli-1 alterations. However, rearrangements of these last two genes are very often associated within the same tumor. Moreover, patterns of coordinated expression of critical cell growth-regulating genes are consistently associated with specific tumor types. These data suggest that Cas-Br-E can induce two types of hematopoietic neoplasias by different mechanisms.
Animals↗
Analysis of proviruses integrated in Fli-1 and Evi-1 regions in Cas-Br-E MuLV-induced non-T-, non-B-cell leukemias.
The DNAs of the Cas-Br-E MuLV-induced leukemias always contain somatically acquired mink cell focus-forming (MCF) recombinant proviruses. MCF recombinants could be involved during leukemogenesis at both preleukemic times and in late-stage tumors. Among the Cas-Br-E-induced non-T-, non-B-cell leukemias, viral integrations were found in the Fli-1 and Evi-1 region in 71% (36 out of 51) and 22% (16 out of 72) of the tumors analyzed, respectively. As an approach to evaluate the contribution of Cas-Br-E MCF recombinant formation in cis-activation of proto-oncogenes, we analyzed the structure of the Fli-1- and Evi-1-associated proviruses by Southern blot hybridization. In Fli-1, we found that the proviruses, ecotropic as well as MCF, are all integrated within a very short DNA region immediately upstream of the initiator ATG, toward the 3' end of a 5' exon (Ben-David, Giddens, Letwin, and Bernstein, 1991, Genes Dev. 5, 908-918). All proviruses are oriented the same way, in the 5' to 3' transcriptional sense. Both provirus types are able to direct the Fli-1 expression to the same extent presumably via a promoter insertion mechanism. Most of the proviruses had no detectable deletion and contained both 5' and 3' LTR sequences with similar U3 sequences. MCF recombinants did not show any selective advantage over ecotropic proviruses for the Fli-1 locus since the frequency of ecotropic to MCF-recombinant virus at the Fli-1 locus was identical to that observed at any other locus. This suggests that the formation of these MCF recombinants is not essential for activation of Fli-1 and that ecotropic Cas-Br-E already possesses the required sequences for full cis-activation of Fli-1. On the other hand, in Evi-1, there is a strict selection for ecotropic proviruses. Presumably, viral genetic elements outside of the U3 region could be critical for the Evi-1 cis-activation.
Animals↗
Monoclonal 3C6F9 distribution in human breast carcinomas: image cytometry of immunocytochemical assays.
MoAbF9 immunoreactivity was investigated in frozen sections of 123 breast carcinomas using an avidin or streptavidin biotin peroxidase kit. A standardized computer image analysis system was used to evaluate immunostaining. The percent of cell surface staining and mean optical densities were correlated with morphological criteria of prognosis such as tumor size histological grade, blood and lymph invasion and axillary lymph node involvement, with immunoreactivity to other MoAb, i.e. Ki67, anti-RE and anti-RP, anti-p.HER-2/neu and with tumor aneuploidy and AgNORs content in tumor cell nuclei. Despite some heterogeneity, MoAbF9 was reactive with all breast carcinomas tested. The percent of F9 immunostained cell surface and mean optical density increased with Ki67 immunoreactivity, tumor aneuploidy and AgNORs nucleus surface but were independent of p.HER-2/neu oncoprotein distribution and tumor receptor content. These findings suggest that F9 could not only allow detection axillary lymph node micrometastases but also be used as plasmatic marker for tumor recurrence and metastases.
Antibodies, Monoclonal↗