Subtracted cDNA Probes Research Articles

Radiolabeling of Subtracted cDNA Probes by Random Oligonucleotide Extension

In this procedure, synthesis of cDNA is performed in the presence of saturating concentrations of all four dNTPs and trace amounts of a single radiolabeled dNTP. After subtraction hybridization, the enriched single-stranded cDNA is radiolabeled to high specific activity in a second synthetic reaction by extension of random oligonucleotide primers using the Klenow fragment of Escherichia coli DNA Pol I. Because the concentrations of dNTP in the first reaction are nonlimiting, both the amounts and size of cDNA generated are greater than those achieved in standard labeling protocols. The subtractive hybridization step can therefore be performed with higher efficiency. Because the resulting population of cDNA is not vulnerable to radiolytic cleavage, it can be stored indefinitely and radiolabeled to higher specific activity when needed. The protocol works best when the cDNA synthesized in the initial synthetic reaction is full length or close to it. For this reason, synthesis of cDNAs is primed by oligo(dT) rather than random hexanucleotide primers. In contrast, the subsequent radiolabeling reaction is primed by random oligonucleotides, yielding shorter DNA products whose size is ideal for hybridization.

Synthesis of Radiolabeled, Subtracted cDNA Probes Using Oligo(dT) as a Primer

Synthesis of Radiolabeled, Subtracted cDNA Probes Using Oligo(dT) as a Primer

Radiolabeling of Subtracted cDNA Probes by Random Oligonucleotide Extension

In this procedure, synthesis of cDNA is performed in the presence of saturating concentrations of all four dNTPs and trace amounts of a single radiolabeled dNTP. After subtraction hybridization, the enriched single-stranded cDNA is radiolabeled to high specific activity in a second synthetic reaction by extension of random oligonucleotide primers using the Klenow fragment of Escherichia coli DNA Pol I. Because the concentrations of dNTP in the first reaction are nonlimiting, both the amounts and size of cDNA generated are greater than those achieved in standard labeling protocols. The subtractive hybridization step can therefore be performed with higher efficiency. Because the resulting population of cDNA is not vulnerable to radiolytic cleavage, it can be stored indefinitely and radiolabeled to higher specific activity when needed. The protocol works best when the cDNA synthesized in the initial synthetic reaction is full length or close to it. For this reason, synthesis of cDNAs is primed by oligo(dT) rather than random hexanucleotide primers. In contrast, the subsequent radiolabeling reaction is primed by random oligonucleotides, yielding shorter DNA products whose size is ideal for hybridization.

Identification of differentially expressed genes in murine mesothelioma cell lines of differing tumorigenicity using suppression subtractive hybridization.

We have previously prepared two B7-1 transfectant clones (AC29 B7-6 and AC29 B7-7) from the AC29 murine mesothelioma (MM) cell line which displayed markedly different in vivo growth rates and susceptibility to cytotoxic T cell killing. Using suppression subtractive hybridisation (SSH), we searched for factors which may determine the biological distinction seen in these clones. We isolated 19 cDNA clones from two SSH generated libraries by screening using subtracted cDNA probes and characterised them using Northern hybridisation, sequencing, RT-PCR and real-time RT-PCR. The 19 cDNA clones comprised 16 different transcripts of which 15 were identified by homology to known genes and one was novel. Expression of a murine endogenous retroviral (mERV) transcript mERV-AC29 was found in the immunogenic AC29 B7-6 clone and parental AC29 but absent in AC29 B7-7. Real-time RT-PCR was used to confirm that galectin-1, the disintegrin/metalloproteinase MDC9 and ribonucleotide reductase M1 were overexpressed in AC29 B7-7. Our results show that SSH is a powerful method for the identification of genes expressed differentially between phenotypically different tumour cell lines or clones. Characterisation of the role of those identified here will provide useful information in understanding genes responsible for differential tumorigenicity.

Novel odorant-binding proteins expressed in the taste tissue of the fly.

A taste tissue cDNA library of the fleshfly Boettcherisca peregrina was screened with a subtracted cDNA probe enriched with taste-receptor-tissue-specific cDNA. Seven genes were identified with sequence similarity to insect odorant-binding protein (OBP) genes. The predicted amino acid sequences of the genes contain the putative signal peptide sequence at the N-terminal and most of them conserve the six cysteines common to known insect OBPs. These genes show a high degree of sequence divergence with approximately 20% amino acid identity. The most striking feature was that all seven of these genes are expressed mainly in the taste tissues, such as the labellum and tarsus, unlike the known insect OBP genes expressed in olfactory tissue. The predicted amino acid sequences had the highest degree of sequence similarity to the Drosophila melanogaster OBPs named pheromone binding protein-related proteins (PBPRPs). These gene products are here referred to as gustatory PBP-related proteins (GPBPRPs) 1-7. Homologous GPBPRP genes were found also in D. melanogaster by database search and are shown to be expressed in Drosophila taste tissues.

Identification of viral induced genes in Ig+ leucocytes of Japanese flounder Paralichthys olivaceus, by differential hybridisation with subtracted and un-subtracted cDNA probes

Up-regulated genes of leucocytes expressing immunoglobulin (Ig+ leucocytes) of hirame rhabdovirus (HRV)-infected Japanese flounder were identified by differential hybridisation, using subtracted and un-subtracted cDNA probes. Ig+ leucocytes were separated from apparently healthy and HRV-infected Japanese flounder by the magnetic beads antibody method using mouse anti-Japanese flounder Ig monoclonal antibody (mab). A cDNA library was constructed from HRV-infected Japanese flounder leucocytes, and was screened with subtracted cDNA probes enriched in genes up-regulated by HRV infection. Fifty cDNAs were isolated for further analysis. These included cDNAs coding for homologues of interferon-inducible 56K protein (IFI56), Stat3, CEF-10, RGS5, inducible poly(A) binding protein, prolylcarboxylpeptidase, basigin III (Ig superfamily), MUC-18 (Ig superfamily), proteasome-nexin 1 (SERPIN), herpes virus entry mediator (TNFR family), collagenase III, gelatinase-b, megakaryocyte stimulating factor, Rab8-interacting protein, IgM, IgD and 20 unknown cDNA clones. The majority of these identified genes are reported for the first time in fish. From leucocytes mRNA for homologues of IFI56, CEF-10, Stat3, SERPIN and inducible poly (A) binding protein expression was shown to increase following HRV infection.

Identification of differentially expressed genes in the visual structures of brain using high-density cDNA grids

The hybridization patterns of 18,371 high-density-grid-arrayed non-redundant complementary DNA (cDNA) clones were examined using three different sources of cDNA probes. The first set of probes was synthesized from mRNA isolated from visual brain areas MT and V4 of Vervet monkey. The second set of probes was derived from cDNA libraries constructed from two micro dissected sets of layers of the monkey L ateral G eniculate N ucleus layers within the visual pathway, namely the magnocellular and parvocellular layers. The third set of cDNA probes was synthesized from the subtracted fractions of the cDNAs enriched for either the magnocellular or the parvocellular layers of the Lateral Geniculate Nucleus. Software, linked directly to the Genbank database, was developed to aid in the rapid identification of both expressed and differentially expressed genes. Our results indicate that both the cDNA probes synthesized from mRNA and cDNA libraries can identify similar fractions of expressed genes. However, the subtracted cDNA probes improve the efficiency of detection for those genes that are expressed at much lower abundance. Analyses of these results for the differential expression patterns of these genes were validated by semi-quantitative PCR on the DNA derived from the whole tissue cDNA libraries. A list of some known genes that are statistically differentially expressed within the magnocellular layers of the LGN and area MT in the primate visual areas is derived.

Novel embryonic genes are preferentially expressed by autonomously replicating rat embryonic and neointimal smooth muscle cells.

We sought to identify and characterize the expression pattern of genes expressed by smooth muscle cells (SMCs) during periods of self-driven replication during vascular development and after vascular injury. Primary screening of a rat embryonic aortic SMC-specific cDNA library was accomplished with an autonomous embryonic SMC-enriched, nonautonomous adult SMC-subtracted cDNA probe. Positive clones were rescreened in parallel with embryonic SMC-specific and adult SMC-specific cDNA probes. We identified 14 clones that hybridized only with the embryonic cDNA ("emb" clones), 11 of which did not share significant homology with sequences in any of the databases. Five of these novel emb genes (emb7, emb8, emb20, emb37, and emb41) were selectively and only transiently reexpressed in vivo by neointimal SMCs during periods of rapid replication. The emb8:embryonic growth-associated protein (EGAP), which was studied the most extensively, was expressed at high levels by cultured, autonomously replicating embryonic and neointimal SMCs but was detected only at low levels even in mitogenically stimulated adult SMCs. Finally, the administration of antisense EGAP oligonucleotides markedly attenuated embryonic and neointimal SMC replication rates. We suggest that autonomous replication of SMCs may be essential for normal vascular morphogenesis and for the vascular response to injury and that these newly identified "embryonic" genes may be part of the molecular machinery that drives this unique growth phenotype.

Expression of a Solanum tuberosum cyclophilin gene is regulated by fungal infection and abiotic stress conditions

Cyclophilins (CyPs) are ubiquitous proteins with an intrinsic enzymatic activity of peptidyl-prolyl cis- trans isomerase that catalyzes the rotation of X-Pro peptide bonds. These enzymes are believed to play a role in the folding of certain proteins. In addition, CyPs might be important in signal transduction processes. A cDNA library was prepared from potato ( Solanum tuberosusm) tubers infected with the fungus Fusarium solani f. sp eumartii. Using a PCR-amplified subtracted cDNA probe, a clone encoding a cytosolic form of CyP, called StCyP ( S̱ olanum ṯ uberosum CyP), was isolated. Except in tubers, StCyP is expressed at high levels in tissues of healthy potato plants. Northern blot analyses revealed that both wounding and fungal infection increased the level of StCyP mRNA in tubers. However, whereas wounding causes a transient accumulation of StCyP mRNA, fungal infection results in a maintained accumulation of this transcript. StCyP mRNA accumulation is also stimulated by the application of absicic acid (ABA) and methyl jasmonate (MeJA) in tubers. Treatment with fungal elicitor or salicilic acid (SA) has no effect on the level of StCyP mRNA accumulation. Together these results indicate that the observed accumulation of StCyP mRNAs in fungal-infected potato tubers might be a response to the wound produced by the penetration and colonization of the tissue by the pathogen. Furthermore, accumulation of StCyP transcripts was also detected when the potato tubers were exposed to heat-shock treatment. These findings support a role for cyclophilins in the plant response to environmental stresses.

Upregulation of a New Microglial Gene,mrf-1, in Response to Programmed Neuronal Cell Death and Degeneration

Cerebellar granule neurons isolated from postnatal day 7 (P7) rats and grown in normal K+ medium begin to degenerate at approximately 4 d in vitro (DIV) and die. To search for genes upregulated in the process of neuronal cell death, differential hybridization was performed with subtracted cDNA probes and a cDNA library from 5 DIV. One of the genes isolated was microglial response factor-1 (mrf-1), which encoded a sequence of 177 amino acids with a single EF-hand calcium-binding motif. By Northern blots, the transcript was upregulated in cerebellar culture at 4 DIV, peaked at 6 DIV, and decreased at 7 DIV. Upregulation was also found when the apoptosis of granule cells was induced by replacing high K+ medium with normal K+ medium. However, when non-neuronal cells were thoroughly eliminated with aphidicolin, an antimitotic agent, the upregulation at 4-7 DIV did not occur. By immunocytochemistry, MRF-1 was detected at 5 DIV in OX-42-positive cells (microglia), and it exhibited an increase in response to granule cell death. MRF-1 levels in microglia purified from cerebral cortex also upregulated in the presence of 5 DIV granule cells. In the developing cerebellum in vivo, levels of mrf-1 mRNA transiently increased in the early postnatal stages, reaching a peak at P7 when cerebellar neurons and astrocytes undergo extensive apoptosis. In adult brain sections, MRF-1 was detected in the perikarya and processes of ramified/resting microglia, and peripheral motor nerve dissection prominently increased the expression in activated microglia surrounding injured central motoneurons. Therefore, mrf-1 appears to be one of the microglial genes that respond to neuronal cell death and degeneration.

Cloning of cDNA encoding the rice 22 kDa protein of Photosystem II (PSII-S) and analysis of light-induced expression of the gene

Cloning of rice cDNA encoding the chlorophyll-binding 22 kDa protein of Photosystem II (PSII-S) and the light-induced expression of the gene are reported. One of the light-responsive cDNA clones, isolated by screening with a light-specific subtracted cDNA probe, was shown to encode PSII-S of rice. Genomic Southern analysis suggested that the PSII-S gene, psbS, is a single-copy gene in rice. A brief exposure to red light induced a severalfold increase in the steady state level of PSII-S transcripts in etiolated seedlings. The red light effect was reversed by far-red light, suggesting involvement of phytochrome in the PSII-S gene regulation. Prolonged exposure (3 h) to blue light, however, revealed a much stronger effect than red light on the accumulation of PSII-S transcripts in the etiolated seedlings. In dark-adapted green plants, prolonged exposure to blue light induced re-accumulation of transcripts encoding PSII-S, whereas red light had little effect.

Cloning Expressed cDNAS from Defined Chromosomal Regions Using Interspecific Microcell Hybrids and Subtractive Hybridization

Genes expressed from a defined genomic interval can be cloned using interspecific microcell hybrids and subtractive hybridization. This approach involves identifying a pair of interspecific hybrids that contain nearly isogenic fragments of a relevant chromosome, except for a region of nonoverlap encompassing the locus of interest. Radiolabeled cDNAs are synthesized from the hybrid cell line that contains the region of interest and hybridized in solution with an excess of biotinylated mRNA from the microcell hybrid lacking the region of interest. Subtracted cDNA probes enriched for sequences expressed from the nonoverlap region are isolated by the addition of streptavidin followed by organic extraction. These subtracted cDNA probes are used to screen a cDNA library prepared from an appropriate tissue, and candidate cDNAs are mapped by Southern hybridization to determine whether they are encoded within the nonoverlap region. This technique is an attractive alternative to other methods for identifying genes in a genomic region because it does not require that the gene of interest be localized to a minimal genetic interval, and the genomic DNA encompassing the region need not be cloned. Additionally, the method detects only expressed genes, simplifying the search for candidate genes within a genomic region.

Isolation of differentially expressed cDNA clones from salt-adapted Aspergillus nidulans.

Differentially expressed cDNA clones were isolated from salt-adapted Aspergillus nidulans (FGSC #359). Poly (A)+ RNA from adapted mycelia was used to construct a lambda Uni-ZAP cDNA library. The library was screened with mixed subtracted cDNA probes. Three-hundred and fifty-seven positive plaques were isolated in the primary screening. Sixty-two randomly selected plaques were purified and placed into eight different cross-hybridization groups. A representative cDNA from each group was used to study expression under unadapted, salt-adapted and salt-shock conditions. These clones, representing eight different genes, displayed enhanced expression under salt stress. Exploratory nucleotide sequencing was performed, and the predicted amino-acid sequence was compared with known gene sequences in the data-bank. Five of the cDNA clones were identified as a mitochondrial (mt) ATPase beta subunit, a mt ATPase subunit 9, a mt transport protein, a ubiquitin-extension protein and a ribosomal protein. Three cDNA clones could not be identified due to lack of adequate homology with known sequences. These results suggest that at least five genes with known function in cellular processes like ATP generation and protein synthesis, and three other genes of unknown identity, are greatly induced in salt-adapted conditions.

A Tetraspan Membrane Glycoprotein Produced in the Human Intestinal Epithelium and Liver That Can Regulate Cell Density-dependent Proliferation

The human cell line HT-29 provides a model system for studying regulation of proliferation and differentiation in intestinal epithelial cell lineages: (i) HT-29 cells cultured in glucose resemble undifferentiated multipotent transit cells located in the lower half of intestinal crypts; (ii) proliferating HT-29 cells cultured in inosine resemble committed cells located in the upper half of the crypt; (iii) nonproliferating, confluent HT-29-inosine cells have features of differentiated enterocytes and goblet cells that overlie small intestinal villi. A cDNA library prepared from HT-29-inosine cells was screened with a series of subtracted cDNA probes to identify proteins that regulate proliferation/differentiation along the crypt-villus axis. A cDNA was recovered that encodes a 202-amino acid protein with four predicted membrane spanning domains and two potential sites for N-linked glycosylation. Levels of this new member of the superfamily of tetraspan membrane proteins (TMPs) increase dramatically as nondividing epithelial cells exit the proliferative compartment of the crypt-villus unit and migrate onto the villus. The protein is also produced in nondividing hepatocytes that have the greatest proliferative potential within liver acini. Three sets of observations indicate that in the appropriate cellular context, intestinal and liver (il)-TMP can mediate density-associated inhibition of proliferation. (i) Accumulation of il-TMP glycoforms precedes terminal differentiation of HT-29-inosine cells and occurs as they undergo density-dependent cessation of growth. il-TMP levels are lower and glycosylation less extensive in HT-29-glucose cells, which do not undergo growth arrest at confluence. (ii) HeLa cells normally do not produce il-TMP. Forced expression of il-TMP inhibits proliferation as cells approach confluence. The extent of il-TMP glycosylation in the transfected cells is similar to that observed in HT-29-inosine cells and greater than in HT-29-glucose cells. (iii) SW480 cells are derived from a human colon adenocarcinoma and do not express il-TMP. Like nontransfected HeLa cells, they do not stop dividing at confluence, whether grown in medium containing glucose or inosine. Expression of il-TMP has no effect on the growth properties of SW480 cells. The extent of il-TMP glycosylation in SW480-glucose cells is similar to that noted in HT-29-glucose cells, lending further support to the notion that il-TMP's activity is related to its state of N-glycosylation.

Cloning and Expression of Glucocorticoid-induced Genes in Fetal Rat Lung Fibroblasts: TRANSFORMING GROWTH FACTOR-β3

Glucocorticoids have been shown to accelerate fetal lung type II cell maturation, and this effect appears, in part, to be mediated via fibroblasts. To identify glucocorticoid induced genes in fetal lung fibroblasts, we screened a cDNA library from cortisol-treated fetal lung fibroblasts with a subtracted cDNA probe which was enriched for sequences specific for cortisol-treated fetal lung fibroblasts. Fifty-seven clones were isolated from the cDNA library. One cDNA represented approximately 30% of the 57 clones. Analysis of DNA sequence homology suggested that this cDNA encodes the rat transforming growth factor-beta 3 (TGF beta 3). We found that TGF beta 3 mRNA was expressed in fetal lung fibroblasts but not epithelial cells. Expression of message in fetal lung fibroblasts was developmentally regulated. TGF beta 3 mRNA levels were low during the pseudoglandular stage (day 18), peaked during the early canalicular stage of lung development (day 19), then fell again at days 20 and 21 (term = 22 days). Exposure of fetal lung fibroblasts to cortisol increased TGF beta 3 mRNA expression in a time- and dose-dependent manner. Maternal administration of dexamethasone also enhanced mRNA expression of TGF beta 3 in fetal lung fibroblasts. These data suggest that glucocorticoids may mediate their stimulatory effect on lung maturation by inducing TGF beta 3 expression in fetal lung fibroblasts.

Cloning and expression of a neural differentiation-associated gene, p205, in the embryonal carcinoma cell line P19 and in the developing mouse

Mouse P19 embryonal carcinoma cells can be reproducibly differentiated into neurons and glial cells upon treatment with high concentration of retinoic acid (RA). In order to understand the molecular mechanisms that control early neural differentiation, we screened a cDNA library made from 24-h RA-treated P19 cells with subtracted cDNA probes. One clone was positive in the secondary screening and was designated as p205. This clone (1.1 kb) has an open reading frame of 317 amino acids with homology to G-protein β subunit. This protein sequence was identical to chicken and human genes previously identified as a major histocompatibility complex-associated gene. The complete conservation of its amino acid sequence between mouse, human and chicken provides strong evidence that the p205 protein fulfills a fundamental function. Developmental Northern blot analysis revealed that a p205 mRNA is expressed at high levels in the embryonic mouse brain, decreasing as development proceeds. In situ hybridization revealed that p205 mRNA is strongly and ubiquitously expressed in the embryonic and early postnatal mouse brain. This expression decreased during postnatal development and was localized in the dentate gyrus, habenula, piriform cortex, paraventricular nucleus of the hypothalamus and supraoptic nucleus of the adult brain. These results suggest that this protein plays an important role in the developing brain and neuronal differentiation.

Characterization of a 7.5-kDa Protein Kinase C Substrate (RC3 Protein, Neurogranin) from Rat Brain

A 7.5-kDa heat- and acid-stable rat brain protein kinase C (PKC) substrate was purified to near homogeneity by a two-step procedure using DEAE-cellulose and bydroxylapatite column chromatography. This 78-amino-acid protein has a sequence identical to that deduced from rat brain RC3 cDNA identified with a cortex-minus-cerebellum subtracted cDNA probe (J. B. Watson et al., J. Neurosci. Res. 26, 397-408, 1990) and exhibits extensive sequence identity to bovine brain neurogranin (J. Baudier et al., J. Biol. Chem. 266, 229-237, 1991). On sodium dodecyl sulfate-polyacrylamide gel electrophoresis this protein, RC3, migrated as a M r 15-18K species in the presence of reducing agent and as heterogeneous species of M r 13-28K in the absence of reducing agent. Phosphorylation of RC3 by PKC α, β, or γ was stimulated by Ca 2+, phospholipid, and diacyiglycerol. A single site, Ser 36, which is adjacent to the predicted calmodulin (CaM)-binding domain, was phosphorylated by these enzymes. Phosphorylation of RC3 by PKC or PKM, a protease-degraded PKC, was inhibited by CaM. The effect of CaM apparently targets at RC3, as phosphorylation of protamine sulfate by PKM was not inhibited by CaM. In the absence of Ca 2+, RC3 formed a stoichiometric complex with CaM as evidenced by an increase in the M r determined by gel filtration chromatography. In the presence of Ca 2+, the affinity of RC3 toward CaM is greatly reduced and Ca 2+/CaM becomes less inhibitory of the PKM-catalyzed phosphorylation of RC3. Phosphorylation of RC3 by PKM prevented the interaction of this protein with CaM even in the absence of Ca 2+. A 20-amino-acid synthetic peptide (AS-20 F-W) containing the PKC phosphorylation site and CaM-binding domain of RC3 (Ala 29-Ser 48) with a substitution of Phe 37 with tryptophan was used to monitor the interaction of this peptide with CaM by spectrofluorometry. In the absence of Ca 2+, CaM caused negligible change in tryptophan fluorescence of the peptide; however, an enhancement and blue-shift of the emission fluorescence was observed in the presence of Ca 2+. It seems that this synthetic peptide, as well as RC3 holoprotein, interacts with CaM through electrostatic interaction in the absence of Ca 2+ but through hydrophobic interaction in the presence of Ca 2+. In rat brain homogenate, RC3 formed a stable complex with CaM in the presence of EGTA, but not in the presence of Ca 2+, as demonstrated by immunoblot analysis following gel filtration chromatography. These results demonstrate that rat brain RC3 is a specific high affinity CaM-binding protein at low levels of Ca 2+ and the interaction of RC3 with CaM is regulated by Ca 2+ as well as by PKC-catalyzed phosphorylation.

Ezrin and Osteonectin, Two Proteins Associated with Cell Shape and Growth, Are Enriched in the Locus Coeruleus

In an attempt to characterize proteins which are enriched or specifically expressed in the locus coeruleus (LC), a region of the brain which plays a critical role in opiate dependence and withdrawal, we have screened a bovine LC cDNA library with an LC minus cerebellum subtracted cDNA probe and isolated several (38) positively hybridizing clones. DNA sequence analysis revealed that two of the clones encoded ezrin and osteonectin, proteins normally associated with cell growth and morphology in peripheral tissues. Regional northern blots from bovine brain and in situ hybridization studies in rat show that ezrin is expressed at high levels in the LC with only low levels detectable in other brain regions. Osteonectin is also abundant in the LC, but in contrast to ezrin, is expressed at high levels in the dorsal raphe and substantia nigra. The results indicate that two proteins, ezrin and osteonectin, are highly enriched in the LC. This raises the possibility that these polypeptides, which play a role in the morphological response of some non-neuronal cell types to growth factors and to other intracellular signals, may also regulate these properties in noradrenergic and other selected neurons in the brain.

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 mitochodnria-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.

Isolation and Characterization of cDNA Clones for Castration-induced mRNAs in the Rat Ventral Prostate.

To identify gene products involved in castration-induced involution of the rat ventral prostate, we constructed a subtraction cDNA library of the ventral prostate from rats castrated for 48 h. The library was screened with subtracted cDNA probes enriched for sequences with a low copy number expressed in intact or castrated rats. As a result of differential screening, 48 cDNA clones representing 10 different induced mRNAs were isolated. The time course of these mRNA inductions after castration was examined. Within the first 24 h after castration, the level of mRNAs for these cDNA clones was significantly increased and it reached its peak by 48-72 h after castration. Although mRNAs for these cDNA clones were expressed in various tissues from intact rats, an increase in mRNA as a response to castration was observed only in the ventral prostate. Partial sequence analyses of the 10 cDNA clones indicate that three cDNA clones represent rat glutathione S-transferase Yb-1, Yb-2 and Yb-3 subunit mRNA sequences, but for others respective homologues could not be found in a search of the GenBank database (release 67).