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M O Rotenberg

Publications and source records attributed to M O Rotenberg.

17 recordsLinked to original sources
PubMed2000

Regulation of EPC-1/PEDF in normal human fibroblasts is posttranscriptional.

The EPC-1 (early population doubling level cDNA-1) gene, also known as pigment epithelium-derived factor, encodes a protein belonging to the serine protease inhibitor (serpin) superfamily that has been reported to inhibit angiogenesis and proliferation of several cell types. We have previously reported that the EPC-1 mRNA and the secreted EPC-1 protein are expressed at levels more than 100-fold higher in early passage, G(0), WI-38 cells compared to either proliferating or senescent WI-38 fibroblasts. To examine the molecular mechanisms that regulate changes in EPC-1 gene expression in WI-38 cells, we isolated and characterized the human EPC-1 gene and determined the mRNA cap site. Transcriptional assays showed no change in the transcription rates of EPC-1 between young proliferating, quiescent, and senescent WI-38 cells. These results suggest posttranscriptional regulation of the EPC-1 gene. Reverse transcriptase polymerase chain reaction measurements (of hnRNA) indicate regulation at the hnRNA level. The regulation of the EPC-1 gene at the level of hnRNA can explain the observed slow increase in the steady-state EPC-1 mRNA levels when cells become quiescent. The reduction of EPC-1 mRNA levels that occurs when cells exit G(0) and are induced to proliferate can be accounted for by a reduction of the EPC-1 mRNA stability in stimulated cells as compared to quiescent cells.

Animals↗
PubMed1998

Senescent WI-38 fibroblasts overexpress LPC-1, a putative transmembrane shock protein.

We have recently reported the isolation of cDNAs for a number of genes that are differentially expressed between nonproliferating early (young) and late (senescent) population doubling level (PDL) WI-38 human, fetal lung-derived, fibroblast-like cells. We now demonstrate that one of these isolates, LPC-1 (Late PDL cDNA-1), derives from an approximately 2.9-kb mRNA species that is expressed at a two- to fivefold higher level in serum-starved, confluent, senescent versus similarly treated young WI-38 cells. Nucleotide sequence analysis of this cDNA confirms its identity with that of a cDNA encoding a marker (p63) for the rough endoplasmic recticulum and a related swine hepatic cardiogenic shock protein. We show that LPC-1 expression in early PDL WI-38 cells is strictly cell cycle-regulated and its expression peaks 9-12 h after serum stimulation of G0 cultures. The steady state levels of LPC-1 transcript in early PDL cells preceeding and following its peak expression are low, reflecting basal levels seen in G0 upon removal of serum. Late PDL cells, however, seem to have lost this tight cell cycle regulation seen in early PDL cells and inappropriately express high levels of the transcript after serum stimulation. Specific antiserum detects a protein of approximately 63 kDa by Western analysis and elicits intense cytoplasmic staining of senescent fibroblasts by immunohistochemistry. Related genomic sequences are found in all mammalian species examined as well as in the chicken. These findings are consistent with the hypothesis that senescent WI-38 cells exhibit a state of growth arrest fundamentally distinct from that of quiescent (G0) young cells.

Animals↗
PubMed1997

Cloning of the human twist gene: its expression is retained in adult mesodermally-derived tissues.

We have previously reported on the isolation of several differentially expressed genes derived from young and senescent non growing WI-38 human fetal lung-derived fibroblasts. A 0.8-kb cDNA clone, isolate EPC-A2 (early population doubling cDNA-A2), encodes the 3' end of the human homolog of the Twist protein. Twist genes encode basic helix-loop-helix DNA-binding transcription factors that play crucial roles in mesoderm development. Here, we report the cloning and sequencing of the genomic human twist gene. It encodes a protein of 201 amino acids with 96% amino acid sequence identity to mouse Twist, and 100% sequence conservation in the DNA-binding region among all species in which it has been characterized. We further show that expression of human twist is retained in mesodermally-derived tissues and cell lines derived from adult donors.

Adult↗
PubMed1995

Analysis of EPC-1 growth state-dependent expression, specificity, and conservation of related sequences.

The transcript for EPC-1 (early population doubling level (PDL) cDNA-1) is induced under conditions of growth arrest due to density-dependent contact inhibition and/or serum deprivation in early-passage but not in senescent WI-38 fibroblasts. We have characterized the EPC-1 transcript with respect to its cell-cycle regulation, tissue specificity, and interspecies conservation of related genomic sequences. In low density, quiescent (serum-deprived), early-passage fibroblasts that are stimulated to proliferate with fresh serum, steady-state EPC-1 transcript levels are steadily reduced until they reach a basal level approximately 24 h after stimulation. However, when early-passage fibroblasts are made quiescent by both serum deprivation and density-dependent contact inhibition and then stimulated with serum, steady-state EPC-1 transcript levels remain relatively constant throughout a 36 h period following serum stimulation. Senescent WI-38 cells (> 95% life span completed) do not express EPC-1 under these conditions. We show that differences in the regulation of EPC-1 transcript levels in early-passage cells are due to differences in growth state rather than changes in cell density or contact. We also show that expression of the EPC-1 transcript is limited to specific cell types and that related genomic sequences are found in all mammalian species examined as well as in the chicken.

Blotting, Northern↗
PubMed1994

Alterations in contact and density-dependent arrest state in senescent WI-38 cells.

Normal human WI-38 fibroblast-like cells in culture undergo a process of senescence, one feature of which is a gradual decline in proliferative capacity. As these cells reach the end of their replicative life span they exhibit decreases in the fraction of cells able to synthesize DNA, in the number of doublings per passage (constant seeding density), and in the cell harvest and saturation densities. They also display increased average cell cycle times, largely at the expense of longer G1 intervals. These alterations are accompanied by morphologic changes, including cell enlargement. Before the end of the replicative life span or phase-out, there is a highly reproducible (55/58 sublines) cell loss of approximately 50%; however, a stable population survives that can exist in a viable yet nonproliferative state for many months. This stable population maintains an extremely low saturation density, representing < 5% of that achieved by early passage cultures. Further, we show that maximum harvest densities achieved by senescent cells are lower, irrespective of seeding densities, i.e. when placed at cell densities higher than those normally achieved by senescent cultures they display a net decline in cell number. This decline continues until the cell density approximates the density that would have been achieved had the cultures been seeded at standard density (1 x 10(4) cells/cm2).(ABSTRACT TRUNCATED AT 250 WORDS)

Biomarkers↗
PubMed1993

Senescent WI-38 cells fail to express EPC-1, a gene induced in young cells upon entry into the G0 state.

Recently we reported the isolation of cDNAs for a number of genes that are differentially expressed between nonproliferating early (young) and late (senescent) population doubling level (PDL) WI-38 human, fetal lung-derived, fibroblast-like cells. We now demonstrate that one of these isolates, EPC-1 (early PDL cDNA-1), derives from an approximately 1.4-kilobase mRNA species that is expressed at a > or = 100-fold higher level in serum-starved, confluent, young versus similarly treated senescent WI-38 cells. Complete nucleotide sequence analysis of this cDNA confirms its identity with that of a cDNA encoding a secreted, retinal pigmented epithelium differentiation factor as well as similarity of the encoded protein with a number of mammalian serine protease inhibitors. We show that EPC-1 expression is associated with G0 growth arrest in WI-38 cells. The mRNA readily accumulates in density-arrested and/or serum-starved young cells but not in log phase, low density young cells. In contrast, EPC-1 transcript abundance and expression of the encoded, secreted protein are both negligible in senescent WI-38 cells under all culture conditions tested. These findings support the hypothesis that senescent WI-38 cells exhibit a state of growth arrest fundamentally distinct from that of quiescent (G0) young cells.

Amino Acid Sequence↗
PubMed1992

Differential gene expression between young and senescent, quiescent WI-38 cells.

To investigate age-related changes in gene expression in WI-38 cells, we isolated RNA from young and senescent, quiescent cultures and made subtracted cDNA libraries. Density-arrested cells were incubated in serum-free MCDB-104 for 3 days. RNA was then isolated and subtracted cDNA libraries were made in the phagemid vector pCDM8. Both by picking clones at random from these subtracted libraries and by differential hybridization screening with subtracted cDNA probes from young and senescent cells, we have identified a total of 11 genes for which RNA is expressed differentially in these quiescent young and senescent WI-38 cultures. Two genes, EPC-1 and EPC-A2, with elevated RNA levels in young cells, have sequences which have not previously been identified. Two of the genes with elevated RNA expression in the senescent cells are the mitochondria-coded genes for NADH dehydrogenase subunit 4 and for cytochrome b. We also identified seven other genes with elevated RNA levels in senescent cells. Three of these, LPC-1, LPC-14 and LPC-24, have been partially sequenced and have not previously been identified. These studies show that density-arrested, serum-deprived, quiescent young and senescent cells express a number of genes differentially. These differences are not growth-dependent, but are age-dependent. Our studies also show that the methods employed here, which include careful regulation of the cell cultures and subtraction of the libraries, result in libraries from which differentially expressed genes can be identified, either by random selection or by differential hybridization screening with subtracted probes.

Aging↗
PubMed1991

Characterization of a cDNA-encoding rabbit brain heme oxygenase-2 and identification of a conserved domain among mammalian heme oxygenase isozymes: possible heme-binding site?

A 1.3-kb rat testis cDNA clone for heme oxygenase-2 (HO-2) was used as a Northern blot hybridization probe, and a single homologous mRNA species, of approximately 1.3 kb in rabbit brain and testis was detected. This contrasted with the observation made with rat brain in which two HO-2 transcripts of approximately 1.3 and 1.9 kb were detected. Use of the same rat HO-2 probe to screen a rabbit brain cDNA library in lambda gt11 resulted in the recovery of a single 1.2-kb cDNA clone. This cDNA exhibits 84% overall nucleotide sequence homology with rat HO-2 and encodes a protein of 35,352 Da, displaying 88% amino acid sequence homology with rat testis HO-2. Furthermore, when expressed in Escherichia coli, the rabbit cDNA-encoded protein displays heme oxygenase activity and cross-reactivity with antibody to rat HO-2. Based on findings obtained through Western immunoblot analysis of partially purified HO-2 protein prepared from rabbit testis and brain, the 35- to 36-kDa molecular form appears to be the major HO-2 form detected in the brain, whereas a 42-kDa species is the predominant form observed in rabbit testis. Having deduced the amino acid sequence of rabbit brain HO-2, we provide a comparison of this sequence with those of rat, mouse, and human HO-1 and rat HO-2, and thereby identify a 24-amino-acid-long peptide region which, except for one residue, is identical in all five species of HO-1 and HO-2 compared (96% similarity), and exhibits 100% similarity in predicted secondary structure (for this region) in all five proteins. We propose that this peptide may be important to the heme binding and isomer-specific tetrapyrrole cleavage activities of the heme oxygenase isozymes.

Amino Acid Sequence↗
PubMed1990

Developmental expression of heme oxygenase isozymes in rat brain. Two HO-2 mRNAs are detected.

Blot hybridization of RNA isolated from rat brain revealed the presence of two HO-2 homologous transcripts (1.3 and 1.9 kilobases (kb] at all stages of development ranging from 1 day before birth to adulthood. The level of both HO-2 messages appeared to be developmentally regulated and a gradual increase was observed from prenatal day 1 to adulthood. The two transcripts were highly homologous as assayed through hybridization studies using probes derived from the 5' end, middle, and 3' end of a cloned rat testis HO-2 cDNA. The 1.3-kb mRNA was essentially identical in size to the testis HO-2 cDNA. The message was efficiently translated in the brain, and is believed to encode the HO-2 protein. It seems unlikely that the 1.9-kb species represents a precursor of the 1.3-kb mRNA, as it was also translated in vivo, although less efficiently than the smaller mRNA species. Neither of the two HO-2 mRNA species were induced by bacterial endotoxin. Unlike HO-2, only one HO-1 transcript of approximately 1.8 kb could be detected. This transcript was of very low abundance and was not developmentally regulated, but could be increased by bacterial endotoxin. The product of this induced message, however, was not detectable by Western immunoblot analysis using antibody raised against liver HO-1. An immunoprecipitate could be detected in brain microsomes by radioimmunoassay using the same antibody. This protein, however, exhibited antigenic properties different from that of the purified liver HO-1 or that of spleen microsomal HO-1. Brain heme oxygenase activity correlated well with the amount of immunoreactive HO-2 protein and both reflect the abundance of the 1.3-kb mRNA message over the course of development.

Animals↗
PubMed1990

Isolation, characterization, and expression in Escherichia coli of a cDNA encoding rat heme oxygenase-2.

In a recent study (Cruse, I., and Maines, M.D. (1988) J. Biol. Chem. 263, 3348-3353), we reported the isolation of a small cDNA fragment encoding a portion of heme oxygenase-2 through immunological screening of a rat testis cDNA library in lambda gt11. We have now used this 274-base pair (bp) cDNA fragment as a hybridization probe for rescreening of the same library, and have thereby recovered a number of additional positive isolates. Of these, three candidates of approximately 900, 1100, and 1300 bp, respectively, were subsequently subcloned and sequenced. Although differing in length, the sequences of these clones were found to be otherwise identical. Moreover, the length of isolate 18B, 1284 bp, corresponded well with that of the single mRNA species (approximately 1300-1350 nucleotides) detected through Northern blot hybridization analysis of rat testis total and poly(A)+RNA. This full- or near full-length cDNA encodes a 315-amino acid protein with a molecular weight of 35,757, in good agreement with the 36,000 estimated molecular weight of heme oxygenase-2. When expressed in Escherichia coli, cDNA encodes a protein that cross-reacts with heme oxygenase-2 antiserum (as assayed by Western immunoblotting) and yields high levels of heme oxygenase activity in bacterial soluble cell extracts. Finally, computer analysis of the heme oxygenase-2 cDNA sequence indicates that the predicted amino acid sequence and hydropathy profile of the heme oxygenase-2 protein exhibit similarity with heme oxygenase-1.

Amino Acid Sequence↗
PubMed1989

Characterization of cDNAs of spliced HPV-11 E2 mRNA and other HPV mRNAs recovered via retrovirus-mediated gene transfer.

Human papillomaviruses (HPVs) are associated with hyperproliferations of cutaneous or mucosal epithelium. These viruses cannot be propagated in any cell culture system. Because cloning cDNA copies of HPV mRNAs recovered from human lesions has met with only very limited success, the characterization of HPV mRNAs has been problematic. Using the Moloney murine leukemia virus vector system (C.L. Cepko, B.E. Roberts, and R.C. Mulligan, 1984, Cell 37, 1053-1062), we have recovered cDNAs of spliced E2 mRNAs of human papillomavirus type 11 and additional mRNAs of type 11 and type 18 and determined the utilization of open reading frames (ORFs) in the DNA sequences. The recovery of cDNA copies of messages with splice sites identical to those previously described strongly suggests that the newly characterized splice donors and acceptors are also authentic. The HPV-11 E2 cDNA contains the intact E6 and E7 ORFs and the beginning of the E1 ORF in the first exon, which is then spliced from nt 847 to the second exon at nt 2622, 100 nucleotides upstream from the initiation codon for the E2 ORF. The initiation codon in the E1 ORF is followed by four additional in-frame AUG triplets and an in-frame termination codon positioned 30 nucleotides upstream from the initiation codon for the E2 protein. The authenticity of this putative E2 cDNA was shown by its ability to provide enhancer transactivating activity in chloramphenicol acetyltransferase (CAT) assays in several cell lines. A mutation in the genomic DNA at this splice acceptor site eliminates its activity, demonstrating that the splice is essential for the expression of the E2 protein. We conclude that the translation of the HPV-11 E2 protein requires internal initiation.

Base Sequence↗
PubMed1989

Characterization of rare human papillomavirus type 11 mRNAs coding for regulatory and structural proteins, using the polymerase chain reaction.

Certain human papillomavirus (HPV) types cause warts, dysplasias, and carcinomas of the ano-genital and oral mucosa. Because of the inability to propagate HPVs in cultured cells, the paucity of viral mRNAs in human lesions, and the complexity of alternatively spliced transcripts derived from different promoters, it has not been possible to ascertain the exact structures of the majority of the mRNA species and the proteins encoded. We have adapted the recently developed polymerase chain reaction to amplify cDNAs of rare, type 11 HPV mRNAs isolated from a productively infected human foreskin xenograft in an athymic mouse. The oligonucleotide primers were designed to flank each of the mRNA splice sites previously mapped by electron microscopic analysis of heteroduplexes formed between cloned HPV-11 DNA and viral mRNAs isolated from genital warts. The splice junctions were determined by direct sequencing of the PCR-amplified cDNA products or after the cDNA was cloned into a plasmid vector. We provide the first direct evidence for the existence of rare mRNAs with the potential to encode regulatory proteins that have been hypothesized to exist for HPVs. Depending on the lengths of the upstream exons, the translation frame used and the possibility of internal reinitiation during translation, one pair of mRNAs with the same splice junction could encode the viral DNA copy number modulating protein E1-M, the enhancer repression protein E2-C, or both. A second pair of mRNAs, also with identical splice junctions, encode the enhancer-regulating protein E2; the longer of the two could also encode, in its 5' exon, either or both of the E6 and E7 proteins. Finally, we demonstrate that the doubly spliced late message for the major virion capsid protein L1 also contains the entire coding region for the early E1 E4 protein in the first two exons, with the initiation codon for the L1 protein located precisely at the splice acceptor of the third exon. The potential of this late mRNA to encode both the E1 E4 protein and the capsid protein could contribute to the preponderance of the E4 protein in the lesion.

Base Sequence↗
PubMed1988

Depletion of Saccharomyces cerevisiae ribosomal protein L16 causes a decrease in 60S ribosomal subunits and formation of half-mer polyribosomes.

We constructed yeast strains containing deletion-insertion null alleles of the RPL16A or RPL16B genes encoding the 60S ribosomal subunit protein L16 to determine the role of L16 in the synthesis and function of ribosomes. Strains lacking a functional RPL16A gene grow as rapidly as wild type, whereas those containing a null allele of RPL16B grow more slowly than wild type. RNA analysis using RPL16 probes revealed that both RPL16 genes are transcribed and that RPL16B transcripts accumulate to twice the level of RPL16A transcripts. No evidence was obtained for the occurrence of dosage compensation at the level of RPL16 mRNA accumulation in either mutant. Strains lacking both RPL16 genes are apparently inviable, demonstrating that L16 is an essential yeast ribosomal protein. Introduction of an extra copy of either RPL16 gene into rpl16b mutants restored wild-type growth rates, indicating that the two forms of the L16 protein are interchangeable. rpl16 mutants are deficient in 60S ribosomal subunits relative to 40S subunits. 43S preinitiation complexes accumulate in half-mer polyribosomes in the absence of sufficient 60S subunits. We postulate that the slow-growth phenotype of rpl16 mutants results from the perturbation of initiation of protein synthesis.

Fungal Proteins↗
PubMed1988

Ribosomal protein synthesis is not regulated at the translational level in Saccharomyces cerevisiae: balanced accumulation of ribosomal proteins L16 and rp59 is mediated by turnover of excess protein.

We have investigated the mechanisms whereby equimolar quantities of ribosomal proteins accumulate and assemble into ribosomes of the yeast Saccharomyces cerevisiae. Extra copies of the cry1 or RPL16 genes encoding ribosomal proteins rp59 or L16 were introduced into yeast by transformation. Excess cry1 or RPL16 mRNA accumulated in polyribosomes in these cells and was translated at wild-type rates into rp59 or L16 proteins. These excess proteins were degraded until their levels reached those of other ribosomal proteins. Identical results were obtained when the transcription of RPL16A was rapidly induced using GAL1-RPL16A promoter fusions, including a construct in which the entire RPL16A 5'-noncoding region was replaced with the GAL1 leader sequence. Our results indicate that posttranscriptional expression of the cry1 and RPL16 genes is regulated by turnover of excess proteins rather than autogenous regulation of mRNA splicing or translation. The turnover of excess rp59 or L16 is not affected directly by mutations that inactivate vacuolar hydrolases.

Gene Expression Regulation↗
PubMed1986

Tripartite upstream promoter element essential for expression of Saccharomyces cerevisiae ribosomal protein genes.

To initiate a genetic analysis of yeast ribosomal protein gene promoters, we have constructed a gene fusion between the yeast ribosomal protein gene RP39A and the Escherichia coli lacZ gene. This gene fusion contains approximately 1,030 nucleotides of the 5' flanking region and the first 49 1/3 codons of RP39A fused in frame to a large 3' end fragment of lacZ. Whether it is introduced into yeast cells on a moderately high-copy-number plasmid, or integrated into the yeast genome at the RP39A locus, this RP39A-lacZ gene directs the synthesis of a hybrid transcript which encodes beta-galactosidase activity. Deletions in the 5' flanking region of RP39A-lacZ were constructed by linker insertion and BAL 31 mutagenesis. The expression of the mutant genes in yeast cells was assayed by measuring RP39A-lacZ mRNA and beta-galactosidase levels. By these means we have shown that the sequences between nucleotides -256 and -170 upstream of RP39A are essential for expression of this gene. Three sequence motifs, HOMOL1, RPG, and a T-rich region, which were found in that order 5'----3' upstream of most yeast ribosomal protein genes, were present within this interval. We found that substitution of the CYC1-lacZ upstream activation site with the fragment from nucleotides -298 to -172 upstream of RP39A, containing the HOMOL1-RPG-T-rich motif in that 5'----3' orientation, fully restored expression of the CYC1-lacZ gene. The essentially of HOMOL1, the RPG sequence, and the T-rich region for wild-type levels of expression of RP39A, the conserved location and order of these sequence motifs in yeast ribosomal protein genes, and the ability of a DNA fragment carrying these three sequence elements to substitute for the upstream activation site regions of CYC1 indicate that these three oligonucleotides may be essential to the transcription of yeast ribosomal protein genes.

Base Sequence↗