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Biomedical subjects

Y N Chang

Publications and source records attributed to Y N Chang.

At least 19 recordsLinked to original sources
PubMed2002

Effect of rice--glycerol complex medium on the production of Lovastatin by Monascus ruber.

Response surface methodology (RSM) was employed to study the effect of the composition of the rice-glycerol complex medium on the production of lovastatin (Lvs) by the ascomycete Monascus ruber in mixed solid-liquid (or submerged) cultures at 25 degrees C. Four components (rice powder, peptone, glycerol, glucose) were studied to evaluate the approximate polynomial for all dependent variables, explaining their effects on the production of Lvs. The best composition derived from RSM regression was (in g/L) rice powder 34.4, peptone 10.8,, glucose 129, KNO3 8.0, MgSO4.7H2O 4.0 and glycerol 36.4 mL/L. With this composition, the Lvs production was 157 mg/L after 10 d of cultivation. In comparison with glycerol and glucose, the rice powder becomes a more suitable carbon source and represents a great potential for the production of Lvs.

Glucose↗
PubMed2001

Production of a biopolymer flocculant from Bacillus licheniformis and its flocculation properties.

Bacillus licheniformis CCRC 12826 produced extracellularly an excellent biopolymer flocculant in a large amount when it was grown aerobically in a culture medium containing citric acid, glutamic acid and glycerol as carbon sources. The biopolymer flocculant was an extremely viscous material with a molecular weight over 2 x 10(6) by gel permeation chromatography. It could be easily purified from the culture medium by ethanol precipitation. It was shown to be a homopolymer of glutamic acid by amino acid analysis and thin layer chromatography and presumed to be poly-glutamic acid (PGA). This bioflocculant efficiently flocculated various organic and inorganic suspensions. It flocculated a suspended kaolin suspension without cations, although its flocculating activity was synergistically stimulated by the addition of bivalent or trivalent cations Ca2+, Fe3+ and Al3+. However, the synergistic effects of metal cations were most effective at neutral pH ranges. The comparison of the flocculating activity between the present biopolymer and a commercial lower molecular weight product showed that the biopolymer of the present study had much higher activity. The high productivity and versatile applications of PGA make its development as a new biodegradable, harmless, biopolymer flocculant economical and advantageous.

Aerobiosis↗
PubMed2000

[Fusion and expression of the gene encoding human Mn-SOD to anti-CEA single-chain antibody in Escherichia coli].

The gene encoding human manganese-superoxide dismutase (Mn-SOD) was fused to anti-carcinoembryonic antigen single-chain antibody gene to construct the fusion gene, then was ligated into prokaryotic expression vector pET-22b(+), The fusion gene was expressed in E. coli at high level, accounting for 24% of the total bacteria soluble protein; and was characterized by SDS-PAGE and Western-blot analysis; the expression product had the CEA-binding ability in RIA, and also had the SOD activity by pyrogallol autoxidation assay. So, the Mn-SOD moiety retains substantial enzymatic activity, where the ScFv moiety can deliver the fusion protein to tumor, Mn-SOD is a potential tumor-suppressor gene, maybe the fusion protein can provide a new pathway to tumor therapy.

Amino Acid Sequence↗
PubMed1999

A novel tumor-specific replication-restricted adenoviral vector for gene therapy of hepatocellular carcinoma.

Transducing and distributing a vector throughout a tumor mass are presently insufficient for effective cancer gene therapy. To overcome these difficulties an adenoviral vector was designed that would replicate specifically in tumor cells. This tumor-specific replication-restricted adenoviral (TSRRA) vector was constructed by requiring that the essential E1A gene be expressed from a tumor-specific promoter, namely, the alpha-fetoprotein (AFP) gene promoter. This promoter was chosen since the AFP gene is highly expressed in 70-80% of patients with hepatocellular carcinoma (HCC) but not in normal adults. HCC is one of the major worldwide causes of cancer death. A vector was constructed (AvE1a04i) and demonstrated to replicate in human AFP-producing HCC cell lines. However, little replication was observed in seven other, non-AFP-producing human cell lines, as well as primary cultures of normal human lung epithelial and endothelial cells. In addition, AvE1a04i was shown to prevent tumor growth of an ex vivo-transduced AFP-expressing HCC cell line but not a non-AFP-expressing cell line. Finally, in situ administration of AvE1a04i into preestablished tumors resulted in a greater than 50% long-term survival rate. This novel TSRRA vector for HCC demonstrated both specificity and efficacy in vitro and in vivo.

Adenoviruses, Human↗
PubMed1999

Retroviral-mediated transfer and expression of the multidrug resistance protein 1 gene (MRP1) protect human hematopoietic cells from antineoplastic drugs.

Multidrug resistance protein (MRP1) is a member of the ATP-binding cassette (ABC) transmembrane transporter superfamily that confers multidrug resistance. The transfer and expression of the MRP1 gene in human hematopoietic stem cells may be a useful alternative to multidrug resistance (MDR1) gene transfer for protection from the myelosuppressive effects of chemotherapy in cancer patients. We constructed a gibbon ape leukemia virus packaging cell line (PG13) using the human MRP1 cDNA in a Moloney murine leukemia virus (MoMuLV) backbone containing a modified LTR. This PG13-based cell line, designated MRP1-PG13, produces retroviral vectors bearing the MRP1 gene at a titer of 1.7x10(5) viral particles/ml. Transduction of the human leukemic cell line K562 showed that viral MRP1-PG13 supernatants routinely transfer the MRP1 gene to approximately 35% of target K562 cells, of which at least one third are capable of proliferating in the presence of otherwise toxic concentrations of etoposide. Southern blot analyses indicated that most clones had only one proviral integration. Northern blot analysis of expanded K562 clones showed the presence of a major full-length approximately 8-kb MRP1 transcript as well as a minor approximately 6-kb transcript in all clones. Flow cytometric analysis of the producer cells and clones of transduced K562 cells demonstrated significantly increased MRP1 expression in these cells (approximately 30-fold increase). Human bone marrow mononuclear cells and CD34+ cells were also transduced with MRP1-PG13 supernatants on fibronectin-coated culture flasks in the presence of SCF, IL-3, and IL-6. PCR analysis of individual hematopoietic colonies in methylcellulose cultures demonstrated proviral DNA in approximately 10% of unselected human hematopoietic progenitor cells cultured from nonsorted mononuclear cell samples and in up to approximately 75% of progenitors when CD34-enriched cell populations were targeted. To assess functional MRP1 gene expression, normal human hematopoietic progenitors and K562 cells were cultured in methylcellulose assays containing vincristine or etoposide. All transduced samples gave rise to approximately 10% drug-resistant colonies, which were shown to be provirus-positive by PCR. Our studies document the development of an amphotropic MRP1 retroviral vector producer cell line and pave the way for large animal and preclinical studies of chemoprotection by MRP1 gene transfer.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1999

Paclitaxel chemotherapy after autologous stem-cell transplantation and engraftment of hematopoietic cells transduced with a retrovirus containing the multidrug resistance complementary DNA (MDR1) in metastatic breast cancer patients.

The MDR1 multidrug resistance gene confers resistance to natural-product anticancer drugs including paclitaxel. We conducted a clinical gene therapy study to determine whether retroviral-mediated transfer of MDR1 in human hematopoietic cells would result in stable engraftment, and possibly expansion, of cells containing this gene after treatment with myelosuppressive doses of paclitaxel. Patients with metastatic breast cancer who achieved a complete or partial remission after standard chemotherapy were eligible for the study. Hematopoietic stem cells (HSCs) were collected by both peripheral blood apheresis and bone marrow harvest after mobilization with a single dose of cyclophosphamide (4 g/m2) and daily filgrastim therapy (10 microg/kg/day). After enrichment for CD34+ cells, one-third of each collection was incubated ex vivo for 72 h with a replication-incompetent retrovirus containing the MDR1 gene (G1MD) in the presence of stem-cell factor, interleukin 3, and interleukin 6. The remaining CD34+ cells were stored without further manipulation. All of the CD34+ cells were reinfused for hematopoietic rescue after conditioning chemotherapy with ifosfamide, carboplatin, and etoposide regimen. After hematopoietic recovery, patients received six cycles of paclitaxel (175 mg/m2 every 3 weeks). Bone marrow and serial peripheral blood samples were obtained and tested for the presence of the MDR1 transgene using a PCR assay. Six patients were enrolled in the study and four patients received infusion of genetically altered cells. The ex vivo transduction efficiency, estimated by the PCR assay, ranged from 0.1 to 0.5%. Three of the four patients demonstrated engraftment of cells containing the MDR1 transgene. The estimated percentage of granulocytes containing the MDR1 transgene ranged from a maximum of 9% of circulating nucleated cells down to the limit of detection of 0.01%. One patient remained positive for the MDR1 transgene throughout all six cycles of paclitaxel therapy, whereas the other 2 patients showed a decrease in the number of cells containing the transgene to undetectable levels. Despite the low level of engraftment of MDR1-marked cells, a correlation was observed between the relative number of granulocytes containing the MDR1 transgene and the granulocyte nadir after paclitaxel therapy. No adverse reactions to the genetic manipulation procedures were detected. Therefore, engraftment of human HSCs transduced with the MDR1 gene can be achieved. However, the overall transduction efficiency and stable engraftment of gene-modified HSCs must be improved before MDR1 gene therapy and in vivo selection with anticancer drugs can be reliably used to protect cancer patients from drug-related myelosuppression.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

Involvement of p38 mitogen-activated protein kinase signaling pathway in the rapid induction of the 78-kDa glucose-regulated protein in 9L rat brain tumor cells.

We have previously shown that treatment with okadaic acid (OA) followed by heat shock (HS) (termed OA --> HS treatment) leads to rapid transactivation of the 78-kDa glucose-regulated protein gene (grp78) in 9L rat brain tumor cells. A cAMP-responsive element-like (CRE-like, TGACGTGA) promoter sequence and a protein kinase A signaling pathway are involved in this induction, and activation of both CRE binding protein (CREB) and activating transcription factor-2 (ATF-2) is required in the above process. Herein, we report that transactivation of grp78, as well as phosphorylation/activation of ATF-2, can be completely annihilated by SB203580, a highly specific inhibitor of p38 mitogen-activated protein kinase (p38(MAPK)). Activation of p38(MAPK) by OA --> HS is also substantiated by its own phosphorylation as well as the phosphorylation and activation of MAPK activating protein kinase-2 in cells subjected to this treatment. The involvement of p38(MAPK) in the activation of ATF-2, which leads to the transactivation of rat grp78, is confirmed by electrophoretic mobility shift assay using a probe containing the CRE-like sequence as well as by transient transfection assays with a plasmid containing a 710-base pair stretch of the grp78 promoter. Together with our previous studies, these results led us to conclude that phosphorylation/activation of CREB upon OA --> HS treatment is mediated by cAMP-dependent protein kinase, whereas that of ATF-2 is mediated by p38(MAPK). The transcription factors may bind to each other to form heterodimers that in turn transactivate grp78 by binding to the CRE-like element. This suggests that distinct signaling pathways converge on CREB-ATF-2, where each subunit is individually activated by a specific class of protein kinases. This may allow modulation of grp78 transactivation by diverse external stimuli.

Animals↗
PubMed1998

Behavioral and cellular protection of rat dopaminergic neurons by an adenoviral vector encoding glial cell line-derived neurotrophic factor.

Previously, we observed that an adenoviral (Ad) vector encoding human glial cell line-derived neurotrophic factor (GDNF), injected near the rat substantia nigra (SN), protects SN dopaminergic (DA) neuronal soma from 6-hydroxydopamine (6-OHDA)-induced degeneration. In the present study, the effects of Ad GDNF injected into the striatum, the site of DA nerve terminals, were assessed in the same lesion model. So that effects on cell survival could be assessed without relying on DA phenotypic markers, fluorogold (FG) was infused bilaterally into striatae to retrogradely label DA neurons. Ad GDNF or control treatment (Ad mGDNF, encoding a deletion mutant GDNF, Ad lacZ, vehicle, or no injection) was injected unilaterally into the striatum near one FG site. Progressive degeneration of DA neurons was initiated 7 days later by unilateral injection of 6-OHDA at this FG site. At 42 days after 6-OHDA, Ad GDNF prevented the death of 40% of susceptible DA neurons that projected to the lesion site. Ad GDNF prevented the development of behavioral asymmetries which depend on striatal dopamine, including limb use asymmetries during spontaneous movements along vertical surfaces and amphetamine-induced rotation. Both behavioral asymmetries were exhibited by control-treated, lesioned rats. Interestingly, these behavioral protections occurred in the absence of an increase in the density of DA nerve fibers in the striatum of Ad GDNF-treated rats. ELISA measurements of transgene proteins showed that nanogram quantities of GDNF and lacZ transgene were present in the striatum for 7 weeks, and picogram quantities of GDNF in the SN due to retrograde transport of vector and/or transgene protein. These studies demonstrate that Ad GDNF can sustain increased levels of biosynthesized GDNF in the terminal region of DA neurons for at least 7 weeks and that this GDNF slows the degeneration of DA neurons and prevents the appearance of dopamine dependent motor asymmetries in a rat model of Parkinson's disease (PD). GDNF gene therapy targeted to the striatum, a more surgically accessible site than the SN, may be clinically applicable to humans with PD.

Adenoviridae↗
PubMed1998

Role of mitogen-activated protein kinase in taxol-induced apoptosis in human leukemic U937 cells.

The induction of apoptosis by Taxol was investigated in human leukemic U937 cells. Treatment of U937 cells with 20 nM Taxol for 24 h induced apoptosis in 30-40% of cells, which resulted in an 80% growth inhibition 3 days after treatment. Synchronous cells at different cell cycle stages exhibited different sensitivities toward Taxol, and their reversion by certain protein kinase inhibitors was also phase specific. Kinetic studies of cell cycle progress reveal that Taxol accelerates the progression of the cell cycle, which facilitates the process of apoptosis, especially for cells initially in the G1 phase. This acceleration may result from transient activation of p42/ 44 mitogen-activated protein (MAP) kinase, because inhibition of upstream MAP/extracellular signal-regulated kinase kinase (MEK1/2) by PD98059 reversed this effect. However, the delayed S-G2-M-phase progression by PD98059 was insignificant. The results suggest that MAP kinase may not only mediate cell cycle progress but may also participate in the apoptosis pathway for cells originally in S phase.

Antineoplastic Agents, Phytogenic↗
PubMed1997

Dual cytotoxic mechanisms of submicromolar taxol on human leukemia HL-60 cells.

Taxol-induced mitotic block and apoptosis were investigated using taxol-sensitive human leukemia HL-60 cells at submicromolar concentrations of the drug. Cells exposed to either 20 nM taxol for 1 hr or 10 nM taxol for 12 hr were able to resume normal growth, whereas cells exposed to 60 nM taxol for 1 hr or 10 nM taxol for 24 hr failed to proliferate after drug removal. Progressive changes in the percentage of mitotic block and apoptosis induced by these four treatment protocols were monitored continuously for 3-5 days after drug removal. Cells treated with 20 nM taxol for 1 hr showed a mitotic block without a subsequent increase in apoptosis, whereas cells treated with 10 nM taxol for 12 hr showed an increase in apoptotic ratio within several hours without an increase in mitotic block. These results indicate that apoptosis does not necessarily result from mitotic block and that these two phenomena can occur independently of each other. Drug sensitivity at progressive stages of the cell cycle was also investigated. The results showed that, in addition to the cells in G2/M phase, the cells in S phase were also sensitive to the drug, especially to a prolonged treatment. These results suggest that, in HL-60 cells, the apoptotic programs can be initiated in either the G2/M or S phase and represent two different cytotoxic mechanisms of taxol.

Antineoplastic Agents, Phytogenic↗
PubMed1997

Dopaminergic neurons protected from degeneration by GDNF gene therapy.

Glial cell line-derived neurotrophic factor (GDNF) supports growth and survival of dopaminergic (DA) neurons. A replication-defective adenoviral (Ad) vector encoding human GDNF injected near the rat substantia nigra was found to protect DA neurons from the progressive degeneration induced by the neurotoxin 6-hydroxydopamine (6-OHDA) injected into the striatum. Ad GDNF gene therapy reduced loss of DA neurons approximately threefold 6 weeks after 6-OHDA lesion, as compared with no treatment or injection of Ad lacZ or Ad mGDNF (encoding a biologically inactive deletion mutant GDNF). These results suggest that Ad vector-mediated GDNF gene therapy may slow the DA neuronal cell loss in humans with Parkinson's disease.

Adenoviridae↗
PubMed1996

Sequential steps in Tat trans-activation of HIV-1 mediated through cellular DNA, RNA, and protein binding factors.

The regulation of HIV expression is controlled by the activity of the Long Terminal Repeat (LTR). Trans-activation by the virally encoded Tat protein is one of the main mechanisms of LTR activation. Tat binds to its target, TAR RNA, and cellular proteins that bind the LTR, Tat, or TAR RNA are important components of the trans-activation process. We will review the factors that have been characterized for a possible involvement in this mechanism. Whereas LTR binding proteins consist of Sp1 and TBP, a large number of factors that bind TAR RNA have been isolated. We have previously cloned two of them by RNA probe recognition: TRBP and La. We have shown that the in vitro and in vivo binding of TRBP to TAR RNA correlates with a constant expression of the protein during HIV-1 infection. Several proteins that interact with Tat have mainly positive, but some negative, effects on trans-activation. Genetic studies have defined that human chromosome 12 encodes a protein that will allow trans-activation in rodent cells. The binding and the functional data about these proteins suggest sequential steps for the Tat trans-activation mechanism. Each of these intracellular molecular events could be the target for molecular intervention against the virus.

Amino Acid Sequence↗
PubMed1995

Effect of herpes simplex virus thymidine kinase expression levels on ganciclovir-mediated cytotoxicity and the "bystander effect".

Transfer of the herpes simplex virus type-1 thymidine kinase (HSV-tk) gene into tumor cells followed by ganciclovir (GCV) administration, will provide selective tumor cell killing. We studied the effect of herpes simplex virus thymidine kinase (HSV-tk) expression level on the HSV-tk/GCV-mediated "bystander effect." Clones of HSV-tk-transduced rat glioma cells (9L) were isolated that stably expressed with different levels of HSV-tk. All clones studied had similar sensitivity to ganciclovir with IC50 values ranging from 0.45 to 1.3 microM. Within certain enzyme level thresholds, in vitro evaluation of the bystander effect has shown that clones with higher level of HSV-tk expression exhibited a better bystander effect. Interestingly, the bystander effect was observed between different cell types. Both the transduction efficiency and bystander effect are essential factors for the success of the antitumor effect by the HSV-tk/prodrug GCV suicide gene system.

3T3 Cells↗
PubMed1994

Direct interactions between autoantigen La and human immunodeficiency virus leader RNA.

We have characterized the in vivo and in vitro binding of human La protein to the human immunodeficiency virus type 1 (HIV-1) leader RNA, the trans-activation response element (TAR). In immunoprecipitation studies using anti-La serum, La-TAR ribonucleoproteins were recovered from HIV-1-infected lymphocytes. Further characterization of this interaction revealed that La has preference for the TAR stem. However, TAR RNA recognition tolerated changes in the primary sequence of the stem as long as the secondary structure was conserved. This structural aspect of La-TAR recognition was confirmed in competition studies in which certain homopolymers influenced complex formation while other single-stranded and double-stranded RNAs had no effect. Deletion mutants of recombinant La protein were used to demonstrate that the residues responsible for binding to polymerase III precursor transcripts overlapped the binding domain for the TAR leader RNA. This finding of a direct interaction between La and TAR has functional implications for translational regulation of HIV-1 mRNAs as demonstrated in the accompanying report (Y. V. Svitkin, A. Pause, and N. Sonenberg, J. Virol. 68:7001-7007, 1994).

Autoantigens↗
PubMed1993

A human DNA segment encompassing leucine and methionine tRNA pseudogenes localized on chromosome 6.

A human genomic clone, designated LHtlm8, that strongly hybridized to a mammalian leucine tRNA(IAG) probe, was found to encompass a pair of tRNA pseudogenes that are transcribed in a homologous cell extract. A leucine tRNA(AAG) pseudogene (TRLP1) is 2.1-kb upstream and of opposite polarity to a methionine elongator tRNA(CAU) pseudogene (TRMEP1). TRLP1 has three nucleotide variations (97% identity) from its cognate leucine tRNA(IAG), while TRMEP1 has a 78% identity with its cognate tRNA. Similar to a number of other eukaryotic tRNA pseudogenes, presumptive precursor tRNA transcripts are generated from the two pseudogenes in vitro, but possibly due to their aberrant and unstable secondary and tertiary structures, no detectable mature tRNA products are observed. The two tRNA pseudogenes are encompassed within a 9.6-kb EcoRI fragment that has been assigned to the chromosomal locus, 6pter-q13, by Southern blot hybridization of human-rodent somatic cell hybrid DNAs with probes derived from the cloned tRNA pseudogenes and flanking sequences. A 4.4-kb EcoRI fragment also harbored in clone LHtlm8 was mapped to human chromosome 11, suggesting that the two EcoRI fragments were inadvertantly ligated together during construction of the genomic library.

Animals↗
PubMed1993

Identification of a large bent DNA domain and binding sites for serum response factor adjacent to the NFI repeat cluster and enhancer region in the major IE94 promoter from simian cytomegalovirus.

The major immediate-early (MIE) transactivator proteins of cytomegaloviruses (CMV) play a pivotal role in the initiation of virus-host cell interactions. Therefore, cis- and trans-acting factors influencing the expression of these proteins through their upstream promoter-enhancer regions are important determinants of the outcome of virus infection. S1 nuclease analysis and in vitro transcription assays with the MIE (or IE94) transcription unit of simian CMV (SCMV) (Colburn) revealed a single prominent mRNA start site associated with a canonical TATATAA motif. This initiator region lies adjacent to a 2,400-bp 5'-upstream noncoding sequence that encompasses a newly identified 1,000-bp (A+T)-rich segment containing intrinsically bent DNA (domain C), together with the previously described proximal cyclic AMP response element locus (domain A) and a tandemly repeated nuclear factor I binding site cluster (domain B). Deleted MIE reporter gene constructions containing domain A sequences only yield up to 4-fold stronger basal expression in Vero cells than the intact simian virus 40 promoter-enhancer region, and sequences from position -405 to -69 (ENH-A1) added to a minimal heterologous promoter produced a 50-fold increase of basal expression in an enhancer assay. In contrast, neither the nuclear factor I cluster nor the bent DNA region possessed basal enhancer properties and neither significantly modulated the basal activity of the ENH-A1 segment. A second segment of domain A from position -580 to -450 was also found to possess basal enhancer activity in various cell types. This ENH-A2 region contains three copies of a repeated element that includes the 10-bp palindromic sequence CCATATATGG, which resembles the core motif of serum response elements and proved to bind specifically to the cellular nuclear protein serum response transcription factor. Reporter gene constructions containing four tandem copies of these elements displayed up to 13-fold increased basal enhancer activity and 18-fold tetradecanoyl phorbol acetate responsiveness in U937 cells, but an ENH-A2 DNA segment encompassing two of the core serum response transcription factor binding sites failed to respond to serum induction in NIH 3T3 cells. Although there are overall similarities in the organizations of both the MIE enhancers and MIE transcription units among human CMV, SCMV, and murine CMV, the specific arrangements of repetitive motifs are quite different, and the bent DNA and ENH-A2 domains appear to be unique to SCMV.

Base Sequence↗
PubMed1993

The lytic origin of herpesvirus papio is highly homologous to Epstein-Barr virus ori-Lyt: evolutionary conservation of transcriptional activation and replication signals.

Herpesvirus papio (HVP) is a B-lymphotropic baboon virus with an estimated 40% homology to Epstein-Barr virus (EBV). We have cloned and sequenced ori-Lyt of herpesvirus papio and found a striking degree of nucleotide homology (89%) with ori-Lyt of EBV. Transcriptional elements form an integral part of EBV ori-Lyt. The promoter and enhancer domains of EBV ori-Lyt are conserved in herpesvirus papio. The EBV ori-Lyt promoter contains four binding sites for the EBV lytic cycle transactivator Zta, and the enhancer includes one Zta and two Rta response elements. All five of the Zta response elements and one of the Rta motifs are conserved in HVP ori-Lyt, and the HVP DS-L leftward promoter and the enhancer were activated in transient transfection assays by the EBV Zta and Rta transactivators. The EBV ori-Lyt enhancer contains a palindromic sequence, GGTCAGCTGACC, centered on a PvuII restriction site. This sequence, with a single base change, is also present in the HVP ori-Lyt enhancer. DNase I footprinting demonstrated that the PvuII sequence was bound by a protein present in a Raji nuclear extract. Mobility shift and competition assays using oligonucleotide probes identified this sequence as a binding site for the cellular transcription factor MLTF. Mutagenesis of the binding site indicated that MLTF contributes significantly to the constitutive activity of the ori-Lyt enhancer. The high degree of conservation of cis-acting signal sequences in HVP ori-Lyt was further emphasized by the finding that an HVP ori-Lyt-containing plasmid was replicated in Vero cells by a set of cotransfected EBV replication genes. The central domain of EBV ori-Lyt contains two related AT-rich palindromes, one of which is partially duplicated in the HVP sequence. The AT-rich palindromes are functionally important cis-acting motifs. Deletion of these palindromes severely diminished replication of an ori-Lyt target plasmid.

Animals↗