Biomedical subjects

S Lin-Chao

Publications and source records attributed to S Lin-Chao.

At least 37 records · Page 2Linked to original sources

PubMed1994

Effects of nucleotide sequence on the specificity of rne-dependent and RNase E-mediated cleavages of RNA I encoded by the pBR322 plasmid.

RNase E, an endoribonuclease encoded by the Escherichia coli ams/rne/hmp1 locus, cleaves RNA I, an antisense regulator of the replication of ColE1 type plasmids, in a single-stranded region near its 5' end. The rne-3071 mutation prolongs the RNA 1 half-life in cells cultured at an elevated temperature and imparts temperature sensitivity on RNase E isolated from the mutant strain. Here we report the effects of specific sequence changes introduced by site-directed mutagenesis on the location of ribonucleolytic cleavage near the 5' end of pBR322 RNA I in rne-3071 and congenic rne+ E. coli and on cleavage of RNA I by RNase E in vitro. Primer extension analyses showed that the occurrence and position of cleavages in vivo and in vitro are altered highly specifically by sequence changes but that the site of cleavage bears no simple relationship to a particular nucleotide order. Our results do not support either the notion that cleavage by RNase E is determined by a consensus sequence or the contrary view that RNase E is a virtually nonspecific single-stranded endonuclease with a preference for cutting 5' to an AU dinucleotide.

Base Sequence↗

PubMed1993

The Escherichia coli pcnB gene promotes adenylylation of antisense RNAI of ColE1-type plasmids in vivo and degradation of RNAI decay intermediates.

Previous work has shown that RNase E-mediated cleavage of RNAI, an antisense repressor of the replication of ColE1-type plasmids, relieves repression in vivo by endonucleolytically converting RNAI to a rapidly decaying product. We report that mutations in the Escherichia coli pcnB gene result in a 10-fold prolongation of the half-life of RNAI decay intermediates and also of truncated RNAI primary transcripts lacking sites attacked by RNase E. Using Northern blotting, primer extension analysis, [32P]GTP capping of 5'-triphosphate termini, and PCR amplification methods, we show that pcnB-mediated acceleration of RNAI degradation is associated with posttranscriptional 3' addition of adenosine residues in vivo to native and processed forms of RNAI. Accumulation of antisense RNAI decay products in pcnB mutants potentially explains the reduced copy number of ColE1-type plasmids seen in the mutated bacteria.

Adenosine Monophosphate↗

PubMed1993

Characterization of a DNA-damage-recognition protein from F9 teratocarcinoma cells, which is inducible by retinoic acid and cyclic AMP.

A nuclear protein that recognizes u.v.-damaged DNA was detected in extracts from murine F9 embryonic stem cells using a DNA-binding assay. The nuclear-protein-binding activity was increased in cells after treatment with retinoic acid/dibutyryl cyclic AMP (dbcAMP), with optimum induction at 6 days. In vitro treatment of nuclear extracts with agents that affect protein conformation (such as urea, Nonidet P40 and Ca2+) slightly modulated the damage-recognition activity. Furthermore, treatment of nuclear extracts with phosphatase dramatically inhibited the binding activity. In addition, damaged-DNA recognition of the nuclear extracts was effectively inhibited by damaged double- and single-stranded DNA. The expression of the nuclear protein with similar characteristics was abundant in HeLa cells and was increased in drug- or u.v.-resistant cells. The findings suggest that the recognition of a u.v.-DNA adduct is modulated, at least in part, by an activity that is induced during retinoic acid/dbcAMP-induced differentiation. These results also imply that the identified damage-recognition protein may be important for the sensitivity or resistance of mammalian cells to DNA damage.

Animals↗

PubMed1993

The ams-1 and rne-3071 temperature-sensitive mutations in the ams gene are in close proximity to each other and cause substitutions within a domain that resembles a product of the Escherichia coli mre locus.

Two temperature-sensitive mutations, ams-1 and rne-3071, in the ams (altered mRNA stability) gene have been used extensively to investigate the processing and decay of RNA in Escherichia coli. We have sequenced these temperature-sensitive alleles and found that the mutations are separated by only 6 nucleotides and cause conservative amino acid substitutions next to a possible nucleotide-binding site within the N-terminal domain of the Ams protein. Computer analysis revealed that the region altered by the mutations has extensive sequence similarity to a predicted gene product from the mre (murein pathway cluster e) locus of E. coli, which has been implicated previously in determining bacterial cell shape.

Adenosine Triphosphate↗

PubMed1992

A direct-repeat sequence of the human BiP gene is required for A23187-mediated inducibility and an inducible nuclear factor binding.

We have recently isolated a functional promoter encoding the human polypeptide-binding protein (BiP) gene from Burkitt's lymphoma cells by polymerase chain reaction (The EMBL Data Library accession number X59969, 1991). This promoter DNA segment (termed BiP670) was fused to the bacterial chloramphenicol acetyltransferase (CAT) reporter gene and expressed in NIH3T3 cells. BiP670 retains basal and Ca2+ ionophore A23187-inducible activities. Using 5' deletion assay, we found three basal expression elements (BEE) in the BiP670. Removal of the distal BBE (BBE3), which is contained in a segment spanning -368/-170, caused a 50% loss of the basal activity; removal together with the middle BBE (BBE2), which is contained in a segment spanning -170/-107, resulted in a further 30% loss of the activity. Further removal of the proximal BBE (BBE1), which spans -107/-39, abolished greater than 95% of the basal expression. In addition, an A23187-inducible element (AIE) appeared to be associated with the BBE1. At least a six-fold inducibility remained as long as the BiP promoter retained the sequences -107/-39. Using an in vitro gel mobility shift assay, an A23187-inducible nuclear factor (AINF) was detected from NIH3T3 cells. DNA binding competition experiments indicate that the -107/-39 segment contains a sequence motif which interacts with this cellular factor. Further analysis showed that the two direct repeats, ranging -108/-73 and -72/-40, are the target for AINF binding. A 3-4 fold increase of the AINF binding to both repeated sequences was detected from induced cells. Similar results were also demonstrated in HeLa cells, suggesting that transcriptional control of BiP gene expression in mammalian cells is conserved. These findings also imply that the identified nuclear factor may be important in mediating transcriptional activation of the BiP gene.

3T3 Cells↗

PubMed1992

Cloning of a functional Burkitt's lymphoma polypeptide-binding protein/78 kDa glucose-regulated protein (BiP/GRP78) gene promoter by the polymerase chain reaction, and its interaction with inducible cellular factors.

The promoter of the human gene encoding the stress-responsive protein polypeptide-binding protein/78 kDa glucose-regulated protein (BiP/GRP78) was isolated from Burkitt's lymphoma cells by PCR. This promoter DNA segment (termed BiP670) or one of its 5' deletion derivatives was fused to the bacterial chloramphenicol acetyltransferase gene and introduced into HeLa cells for transient expression. BiP670 retained transcriptional activity at both the basal and Ca2+ ionophore A23187-inducible levels. However, there was no significant increase in promoter activity following a 5 h induction with 7 microM-A23187, and less than 5-fold induction at 15 h. In contrast, the steady-state mRNA level was induced by 18-fold at 5 h. The in vivo transactivation assays with BiP670 5' deletion derivatives indicate that the putative A23187-inducible element is located within a 70 bp DNA segment (i.e. spanning -39 to -107 bp upstream of the transcriptional initiation site). Using an in vitro gel mobility shift assay, A23187-inducible nuclear factors were identified from HeLa cell extracts. DNA-binding competition experiments also suggest that the 70 bp DNA segment contains a potential sequence motif for the binding of the A23187-inducible nuclear factors.

Base Sequence↗

PubMed1992

High copy number of the pUC plasmid results from a Rom/Rop-suppressible point mutation in RNA II.

The plasmids pUC18 and pUC19 are pBR322 derivatives that replicate at a copy number several fold higher than the parent during growth of Escherichia coli at 37 degrees C. We show here that the high copy number of pUC plasmids results from a single point mutation in the replication primer, RNA II, and that the phenotypic effects of this mutation can be suppressed by the Rom (RNA one modulator)/Rop protein or by lowering the growth temperature to 30 degrees C. The mutation's effects are enhanced by cell growth at 42 degrees C, at which copy number is further increased. During normal cell growth, the pUC mutation does not affect the length or function of RNA I, the antisense repressor of plasmid DNA replication, but may, as computer analysis suggests, alter the secondary structure of pUC RNA II. We suggest that the pUC mutation impedes interactions between the repressor and the primer by producing a temperature-dependent alteration of the RNA II conformation. The Rom/Rop protein may either promote normal folding of the mutated RNA II or, alternatively, may enable the interaction of sub-optimally folded RNA II with the repressor.

Bacterial Proteins↗

PubMed1992

Overexpression of glutathione S-transferase and elevation of thiol pools in a multidrug-resistant human colon cancer cell line.

A human colon cancer cell line with acquired multidrug resistance (MDR) was assayed for the intracellular GSH level and the activity of GSH-S-transferase (GST), which catalyzes the conjugation reaction of electrophilic drugs with GSH. The GSH level and GST activity (as measured with 1-chloro-2,4-dinitrobenzene) were elevated in the resistant cells by 1.7-fold and 2-fold, respectively. This elevated catalytic activity of the resistant cells was reflected in a 2-fold increase in GST-pi mRNA, which was not the result of gene amplification. In addition, buthionine sulfoximine, a specific inhibitor of GSH synthesis, significantly increased Adriamycin sensitivity in both the MDR and the parental cells, affecting the former more than the latter. The effects seen with buthionine sulfoximine were not seen with puromycin and actinomycin D. A dramatic overexpression of mdr1, a P-glycoprotein gene responsible for the MDR phenotype, was also observed in the MDR cells. In contrast, none of these products (i.e., mdr P-glycoprotein, GSH level, total GST activity, GST-pi gene copy, and GST-pi mRNA level) was elevated in HeLa cells resistant to cisplatin and some alkylating agents, supporting the notion that the acquisition of cisplatin resistance differs from the mechanism of MDR. These results indicate that the intrinsic GSH level and GST-pi activity affect anthracycline resistance per se and not MDR in the human colon cancer cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1991

Ca(2+)-mediated inhibition of a nuclear protein that recognizes UV-damaged DNA and is constitutively overexpressed in resistant human cells: DNA-binding assay.

A nuclear protein that recognizes UV-damaged DNA was detected from HeLa cells using DNA-binding assay. Treatment of cells with Ca2+ ionophore (A23187) caused a dramatic inhibition of the damage-recognition activity. In contrast, in vitro treatment of nuclear extracts with agents that affect protein conformation (such as urea, NP40 and Ca2+) did not significantly affect on the damage-recognition activity. The Ca(2+)-mediated inhibition of UV damage recognition was reconstituted by the addition of the cytosolic extracts, suggesting that the Ca2+ effect does not directly act on the UV damage-recognition protein. The expression of the detected nuclear protein was increased in UV-resistant HeLa cells. In contrast, the level of this protein was dramatically reduced in UV-sensitive xeroderma pigmentosum group A cells. In addition, UV damage-recognition protein is resistant to RNase, and is independent of the previously identified proteins that bind cisplatin-DNA adduct. These findings implied that the recognition of UV-DNA adduct is modulated by the intracellular level of Ca2+.

Binding, Competitive↗

PubMed1991

Co-amplification and over-expression of two mdr genes in a multidrug-resistant human colon carcinoma cell line.

The human P-glycoprotein gene family contains the mdr1 and the mdr3 gene. The mdr1 P-glycoprotein is over-expressed in multidrug resistant (MDR) tumor cells and is believed to play a role in the elimination of certain cytotoxic drugs used in the chemotherapy of cancer. The mdr3 gene has not been found to be amplified or over-expressed in MDR cells. In this study, gene-specific mdr gene probes were developed for the detection of the gene and the total mRNA level. Southern and Northern hybridization analyses showed that the mdr genes and the mRNA levels were increased 30--40-fold in a MDR human colon cancer cell line. In addition, this MDR cell line had an altered growth rate and morphology and detectable double minute chromosomes.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1991

Identification of inducible damage-recognition proteins that are overexpressed in HeLa cells resistant to cis-diamminedichloroplatinum (II).

Two cis-diamminedichloroplatinum (II) (cisplatin)-inducible proteins [b 130 (approximately 130 kDa) and b95 (approximately 95 kDa]) in HeLa cells that recognize both the cisplatin-modified and u.v.-modified DNA were identified in this study. These damage-recognition proteins were overexpressed in cisplatin-resistant HeLa cells. The results suggest that the damage-recognition proteins are regulated in the cells in response to DNA damage, and they may be important for DNA repair and probably the emergence of cisplatin resistance.

Blotting, Southern↗

PubMed1991

The rate of processing and degradation of antisense RNAI regulates the replication of ColE1-type plasmids in vivo.

We show that the rate of degradation of RNAI, an anti-sense repressor of the replication primer RNAII, is a key element of control in the replication of ColE1-type plasmids in vivo. Cleavage of RNAI by RNAase E, a ribosomal RNA-processing enzyme encoded or controlled by the rne (also known as ams) locus, relieves repression by endonucleolytically converting RNAI to a very rapidly decaying product, pRNAI-5. A 5' triphosphate-terminated homolog of pRNAI-5 is degraded slowly and consequently inhibits replication. Nucleotide substitutions within the RNAase E cleavage sequence alter RNAI half-life and plasmid copy number, changing also the incompatibility phenotype. RNAI variants lacking the sequence cleaved by RNAase E are eliminated by growth rate-dependent degradation, resulting in growth-responsive control of plasmid replication and copy number.

Bacteriocin Plasmids↗

PubMed1991

Enhanced host cell reactivation of damaged plasmid DNA in HeLa cells resistant to cis-diamminedichloroplatinum(II).

Human HeLa cells resistant to cisplatin were established by stepwise selection. The selected cells showed a 15- to 20-fold cisplatin resistance (CPR) at the dose level resulting in 50% inhibition. These cells were cross-resistant to mitomycin C, melphalan, and ethyl methanesulfonate but not to Adriamycin, colchicine, or vinblastine. The expression of cisplatin-damaged plasmid DNA carrying the bacterial chloramphenicol acetyltransferase (CAT) gene after its transfection into CPR cells was enhanced by approximately 3-fold. This did not correlate with the degree of CPR. However, the development of the CPR phenotype paralleled the enhanced CAT activity. The addition of aphidicolin (an inhibitor of DNA alpha-polymerase) to CPR cells effectively diminished the enhanced CAT activity and CPR. These studies have identified an enhanced host cell reactivation of the damaged plasmid in the acquisition of CPR, suggesting that DNA repair is a potential mechanism for the development of CPR phenotype in human cells.

Antineoplastic Agents↗

PubMed1991

Cross-resistance to UV radiation of a cisplatin-resistant human cell line: overexpression of cellular factors that recognize UV-modified DNA.

A human cell line selected for cisplatin resistance (CPR) was irradiated with UV light and showed cross-resistance to UV light. Applying a modified chloramphenicol acetyltransferase assay, we observed that CPR cells acquired enhanced host cell reactivation of a transfected plasmid carrying UV damage. Gel mobility shift analysis indicated that two nuclear factors that recognize UV-modified DNA were overexpressed in CPR cells. In addition, factors that bind UV-modified DNA were independent from the factors that bind cisplatin-modified DNA. The significance of the identified binding factors, possibly DNA repair enzymes, is discussed.

Binding, Competitive↗

PubMed1990

Increased mdr gene expression and decreased drug accumulation in a human colonic cancer cell line resistant to hydrophobic drug.

An adriamycin-resistant human colonic cancer cell line was characterized. This clone exhibits the classical multidrug resistance (MDR) phenotype, being cross-resistant to hydrophobic drugs such as colchicine, and vinblastine. In contrast, this clone shows a normal response to DNA-damaging agents. The appearance of MDR in these cells was linked to a decreased accumulation of the drug [3H]colchicine as compared to the drug-sensitive cells. This MDR line expressed 80-100 fold increased levels of the specific 4.5-kb mdr mRNA, and a gene amplification. Our results indicate that MDR in human colonic cancer cells can result from increased expression of at least one member of the mdr gene family.

Antineoplastic Agents↗

PubMed1990

Rapid two-stage polymerase chain reaction amplification of chromosomal DNA segments in lysates made from monolayer cultures attached to microcarrier beads.

We describe a method for the rapid two-stage amplification and detection by ethidium bromide staining of chromosomal nucleotide (nt) sequences in lysates made directly from anchorage-dependent cells attached to microcarrier beads. The procedure circumvents the need for cell detachment steps prior to analysis, facilitates the collection, transfer, and manipulation of the cells being studied, and makes unnecessary the use of Southern-blot hybridization for identification of specific nt sequences present in a small fraction of cells within a heterogeneous population.

Animals↗

PubMed1990

Coordinated induction of two unrelated glucose-regulated protein genes by a calcium ionophore: human BiP/GRP78 and GAPDH.

The induction of human BiP/GRP78 and GAPDH protein genes by the calcium ionophore A23187 was determined. Steady-state levels of mRNA for both the glucose starvation-responsive BiP/GRP78 gene and the glucose-responsive GAPDH gene were dramatically induced in a variety of human cells. There is a homologous palindromatic sequence GCCGTTAACGGC in the active promoter region of the two genes that is known to be required for the induction of mammalian BiP/GRP78 by A23187. The evidence confirms in general the function of this element in the regulation of calcium-associated gene activity.

Base Sequence↗

PubMed1990

Phenotypic reversion of cisplatin resistance in human cells accompanies reduced host cell reactivation of damaged plasmid.

Revertant cell lines were established from cisplatin (CP) resistant HeLa cells. Expression of CP damaged plasmid DNA carrying bacterial chloramphenicol acetyltransferase (CAT) gene after transfection into cells was measured. Revertant cells showed reduced host cell reactivation of damaged plasmid, as compared to resistant cells. Addition of aphidicolin, an inhibitor for DNA polymerase alpha, to resistant cells effectively blocked enhanced plasmid reactivation and acquired resistance. The results are consistent with the notion that cellular ability in repair CP-damaged DNA is a mechanism for CP resistance.

Aphidicolin↗