Differentiation-defective mutants of mouse embryonal carcinoma cells: response to hexamethylenebisacetamide and retinoic acid

We have generated by mutagenesis eight differentiation-defective sublines from three murine embryonal carcinoma (EC) cell lines. These mutants grossly resemble parental cells in the absence of inducers of differentiation. Based upon response to retinoic acid (RA) or hexamethylenebisacetamide (HMBA), the mutants can be grouped into three types: (a) RA-selected cells that lack cellular RA binding protein (cRABP) activity and fail to differentiate in response to RA or HMBA; (b) RA- or HMBA-selected cells that possess cRABP but differentiate poorly, if at all, in the presence of RA or HMBA; and (c) cells originally selected for lack of response to HMBA but which retain cRABP and the ability to differentiate in response to RA.     

Complementation analyses of differentiation-defective embryonal carcinoma cells

We have generated cell hybrids by fusing embryonal carcinoma (EC) cells which fail to differentiate in response to retinoic acid (RA) and/or hexamethylenebisacetamide (HMBA). The first two classes of hybrids were between an RA- line (also unresponsive to HMBA) that lacks cellular RA binding protein (cRABP) activity and HMBA- lines which possess cRABP and differentiate in the presence of RA. All of the hybrid clones possessed cRABP and differentiated normally upon exposure to either RA or HMBA. When the aforementioned RA- mutant was fused with a second mutant which was refractory to RA and HMBA but possessed cRABP activity, the resultant hybrid clones were responsive to both RA and HMBA and had cRABP activity. These results suggest that all of these mutants were recessive and complementary. Tumors from these hybrid lines differentiated extensively, in some instances much more so than the mutant parental lines and even the wild-type lines from which the mutants were derived. Based upon these observations, we propose that various EC lines might differentiate poorly in tumor form for different reasons. Hybrids between two differentiation-defective, cRABP- lines appeared to be at least partially complemented for responsiveness to RA and HMBA. These hybrids contained low but detectable levels of cRABP. This is not a consequence of tetraploidy since fusions between cells from the same mutant line retained their differentiation-defective phenotype and possessed little or no cRABP activity. Unlike tumors from the other hybrids described above, tumors from these hybrid lines expressed a very restricted pattern of differentiated cell types. This might be because the mutant lines in the latter hybrids originally derived from the same wild-type EC line.     

Isolation and characterization of the cDNAs corresponding to mRNAs abundant in undifferentiated mouse embryonal teratocarcinoma stem cells, but not in differentiated mouse parietal endoderm cells

As retinoic acid (RA) and dibutyryl cAMP (cAMP) treatment induces differentiation of mouse teratocarcinoma F9 cells into parietal endoderm cells in vitro, we initiated studies on the molecular mechanisms underlying early mammalian cell differentiation in this system. We constructed cDNA libraries on the poly(A)+RNAs extracted from the undifferentiated F9 cells, and screened for cDNA sequences expressed abundantly in F9 cells, but not in terminally differentiated mouse parietal endoderm PYS-2 cells. Six different cDNA clones were isolated and characterized. The levels of RNAs hybridizable to these clones were at most 5 to 24% in the PYS-2 cells when compared with those in the undifferentiated F9 cells. The six clones were classified into two groups on the basis of their responses to the RA and cAMP treatment. In F9 cells, the levels of RNAs hybridizable to the first group, which contained four clones, were decreased within 72 h after the addition of RA and cAMP, while those of the second group, which contained the remaining two clones, did not decrease significantly. One of the first group clones, named pF9-1, corresponded to the mouse "early transposon-like elements" and another, named pF9-4, hybridized to multi-size RNAs extracted from the undifferentiated F9 cells. The mouse genomic DNA sequences hybridizable to pF9-4 were repeated approximately 5,000 times, and comprise a new gene family, the expression of which is developmentally regulated in mouse F9 cells.     

Sodium butyrate induces histone hyperacetylation and differentiation of murine embryonal carcinoma cells

Cells from embryonal carcinoma (EC) lines 6050AJ and PCC4.aza 1R differentiate in response to treatment with sodium butyrate as well as retinoic acid (RA) or hexamethylenebisacetamide (HMBA). Murine 6050AJ EC cells exposed to sodium butyrate possess hyperacetylated forms of histones H4 and altered forms of histones H2a and H2b, whereas histones from cells treated with other inducers appear to be unaffected. These results might indicate that the mechanism by which sodium butyrate promotes differentiation of EC cells is different from the ways in which RA and HMBA act. Differentiation-defective PCC4(RA)-1 EC cells fail to respond to RA, presumably because they possess minimal amounts of active binding protein for RA (cRABP). Sodium butyrate treatment of these cells results in only a modest level of differentiation. On the other hand, exposure to sodium butyrate plus RA leads to extensive differentiation. As is the case with 6050AJ cells, PCC4(RA)-1 cells treated with sodium butyrate also contain hyperacetylated histones. Furthermore, these cells now possess high levels of cRABP. The latter observations suggest that sodium butyrate has the ability to reactivate a silent cRABP gene in PCC4(RA)-1 cells and thereby lead to extensive differentiation via the retinoid pathway when RA is added.     

Molecular cloning of cDNA corresponding to mRNA species whose steady state levels in the thyroid are enhanced by thyrotropin. Homology of one of these sequences with ferritin H

A lambda gt11 cDNA library was constructed using poly(A)+ mRNA from thyrotropin (TSH)-stimulated Fisher rat thyroid (FRTL5) cells. The library was screened for nonthyroglobulin cDNA sequences by differential plaque filter hybridization using single-stranded cDNA probes synthesized from mRNA prepared from quiescent and TSH-stimulated FRTL5 cells. Thyroglobulin cDNA-containing recombinants in the library were avoided by prehybridizing the TSH probe to excess thyroglobulin cDNA. Of 48,000 clones screened, 60 were chosen as representing mRNA species whose abundance was increased in TSH-stimulated versus quiescent cultures. Southern blot analysis of 9 clones confirmed that the TSH-cDNA probe hybridized to a greater extent to the cDNA inserts than did the control probe. cDNA insert sizes varied between 0.3 kilobase (kb) and 1.0 kb. Northern slot blot analysis using as probes the cDNA of four of these clones (FC4, FC26, FC29, and FC43) demonstrated that TSH stimulation of FRTL5 cells increased the steady state levels of the respective mRNA species by 4-12-fold. For all 4 clones, increases in mRNA levels were apparent within approximately 1 h and were maximal after 14-18 h of TSH stimulation. Determination of the partial nucleotide sequence of these 4 clones confirmed that none was thyroglobulin, thyroid peroxidase, or any other gene previously reported to be stimulated by TSH. Three of the clones bore no homology to any known nucleotide sequence, but FC26 was 85% homologous with human ferritin H. Northern blot analysis using the FC26 cDNA insert as a probe confirmed hybridization to an mRNA species of 1 kb, the known size of ferritin H mRNA. In summary, using the technique of differential plaque filter hybridization, we have identified 4 new genes whose mRNA levels are increased by TSH stimulation of thyroid cells. One of these genes is homologous to human ferritin H.     

Isolation of differentially expressed human cDNA clones: similarities between mouse and human embryonal carcinoma cell differentiation

The study of early human development is of great importance but has been limited by the lack of suitable reagents. Recently, however, the human embryonal carcinoma (EC) cell line NT2D1 has been isolated. This cell line will differentiate upon exposure to retinoic acid (RA). A cDNA library was constructed from poly(A)+ RNA derived from NT2D1 cells treated with 10(-5) M-RA for 7 days (delta NT2D1 cells). By differential cDNA screening, it was found that 1.12% of delta NT2D1 cDNA recombinants screened detected an increase in signal with 32P-cDNAs derived from delta NT2D1 as compared with NT2D1. To compare RA-induced differentiation of mouse and human EC cells, the delta NT2D1 cDNA library was rescreened with 32P-cDNAs derived from the mouse EC cell line F9 and the result compared with 32P-cDNA derived from F9 differentiated to parietalendoderm (F9PE)-like cells and visceral-endoderm (F9VE)-like cells. Approximately 1.2% of the delta NT2D1 cDNA recombinants detected a differential increase in signal following differentiation of mouse EC cells to F9VE and/or F9PE. Of these homologous regulated sequences, 0.3% were common to both mouse and human EC cell RA-induced differentiation. Five different cDNA clones were isolated that detect a marked increase (5- to 75-fold) in mRNA abundance following RA-induced differentiation of NT2D1. Of these five clones, three detect homologous mRNAs which also increase in abundance following differentiation of the mouse EC cell line F9 to PE- and/or VE-like cells; the other two clones do not detect sequences in the mouse mRNAs tested. One clone shows homology to SPARC, a gene known to be regulated during mouse embryonic development. While another clone, SO5A, has a limited range of expression, being detected in F9VE and in a human parietal-endoderm-like cell, but not in F9PE and a human visceral-endoderm-like cell. This work shows that there are both similarities and differences in mouse and human EC cell differentiation, and these cDNA clones provide some of the first reagents for studying the molecular biology of human development.     

Efficient isolation of cDNA clones encoding rheumatoid arthritis autoantigens by lambda phage surface display

Bacteriophage lambda surface display was used to isolate cDNA clones encoding autoantigens recognized by synovial fluid (SF) or sera from patients with rheumatoid arthritis (RA). We constructed cDNA libraries from human synovial sarcoma cells and synovial tissue, using the surface display vector lambdafoo. The cDNA libraries were screened by affinity selection using 40 SF and 44 sera as probes separately immobilized in microtiter wells. Phage clones isolated encode 13 different autoantigens; an unknown protein, two proteins previously unanalyzed as autoimmune antigens, three proteins previously unknown to be recognized by RA sera, and seven known RA antigens. When analyzed their sensitivity and specificity for RA by phage enzyme-linked immunosorbent assay, frequencies of sera that recognize the newly-isolated autoantigens ranged from 20.5 to 6.8% of a panel of RA sera, and 13.6-0% of other autoimmune disease sera. These results indicate that the lambda phage surface display may be powerful for the isolation of cDNA clones encoding autoantigens recognized by SF or sera from patients with not only RA but also other autoimmune diseases.     

Mouse mammary tumor virus-related sequences in mouse lymphocytes are inducible by 12-O-tetradecanoyl phorbol-13-acetate

cDNA libraries from EL-4 cells treated with 12-O-tetradecanoyl phorbol-13-acetate (TPA) were screened for TPA-inducible sequences by differential hybridization. The most abundant inducible species was a sequence similar to that of mouse mammary tumor virus (MMTV). Induction of the mRNA corresponding to the MMTV-related sequences was already evident 30 min after TPA treatment, whereas the maximum accumulation occurred after 20 h of exposure to TPA. TPA also increased levels of MMTV-related RNA in the normal spleen cells of BALB/c and C57BL/6 mice. The level of RNA expression corresponding to MMTV-related sequences, however, was markedly elevated in EL-4 cells as compared with normal spleen cells. Southern blots of EL-4 cell DNA showed that the MMTV-related sequences were inserted into multiple locations of the EL-4 genome. Sequence analysis revealed that the MMTV-related cDNA clones included a part of the env gene and the right long terminal repeat of MMTV. However, the cDNA sequences were substantially different from published MMTV proviral sequences, most notably because of a contiguous deletion of 491 base pairs in the open reading frame within the U3 region.     

A survey of genes in the Atlantic salmon (Salmo salar) as identified by expressed sequence tags

We describe the construction and quality analysis of six cDNA libraries from the liver, ovary, testis, brain, spleen and muscle tissues of adult Atlantic salmon. The cDNA libraries were then screened with total cDNA probes to catalogue clones representing the abundant and rare mRNA populations in each tissue. Subsequently, the 5'-terminal DNA sequences of 1152 cDNA clones, composed of 96 clones from each of the abundant and rare mRNA populations in the six tissues, were determined. Bioinformatic analysis revealed that 510 (50%) of the salmon expressed sequence tags (ESTs) of sufficient length showed significant homology to previously identified genes from salmonid and other species, while 517 (50%) of salmon ESTs were unidentified or novel. After accounting for multi-EST redundancy, the 510 identified ESTs provided DNA sequence markers for 178 salmon genes which are listed in terms of tissue of origin and mRNA abundance class.     

H3 and H4 histone cDNA sequences from Xenopus: a sequence comparison of H4 genes

Ovarian poly (A) + RNA from Xenopus laevis and Xenopus borealis was used to construct two cDNA libraries which were screened for histone sequences. cDNA clones to H4 mRNA were obtained from both species and an H3 cDNA clone from Xenopus laevis. The complete DNA sequences of these clones have been determined and are presented. These new sequences are compared with other H3 and H4 DNA sequences both in the coding and 3' noncoding regions. We find that there is considerable non-random codon usage in ten H4 genes. In addition there are some sequence similarities in the 3' noncoding regions of H3 and H4 genes.     

Human apolipoprotein B: identification of cDNA clones and characterization of mRNA.

Apolipoprotein B (apoB) is a major protein component of low density and very low density lipoproteins. Because of its large size and heterogeneity, molecular studies of apoB have been difficult, and its structure and regulation remain poorly understood. We now report the identification of human apoB cDNA clones by antibody screening of hepatoma libraries in the expression vector lambda gt11. Both oligo(dT) primed and random primed libraries were constructed and screened with polyclonal antibodies to intact apoB, as well as with antibodies raised against a synthetic peptide based on the limited amino acid sequence available for apoB. The identity of the clones was unambiguously established by comparisons of the cloned cDNA sequences with apoB amino acid sequences. The clones hybridize to an exceptionally large 20 kb mRNA that is present in liver and intestine but not other tissues examined, consistent with the distribution expected from protein biosynthetic studies. The properties of the mRNA have implications for the biogenesis of the multiple apoB molecular weight forms secreted by liver and intestine.     

The early genetic response to light in the green unicellular alga Chlamydomonas eugametos grown under light/dark cycles involves genes that represent direct responses to light and photosynthesis

In the green unicellular alga Chlamydomonas eugametos, cellular division is readily synchronized by light/dark cycles. Under these conditions, light initiates photosynthetic growth in daughter cells and begins the G1 phase. Genes whose expression is regulated upon illumination are likely to be important mechanisms controlling cell proliferation. To identify some of those genes, two cDNA libraries were prepared with poly(A)⁺ extracted from cells either stimulated with light for 1 h or held in darkness (quiescent cells) during the same period. To restrict our analysis to those genes that are part of the primary response, cells were incubated in presence of cycloheximide. Differential screening of approximately 40 000 clones in each library revealed 44 clones which hybridize preferentially with a [³²P] cDNA probe derived from RNA of light-stimulated cells and 15 clones which react selectively with a [³²P] cDNA probe synthesized from poly(A)⁺ RNA of quiescent cells. Cross-hybridization of these clones identified 4 independent sequences in the light-induced (LI) collection and 2 in the uninduced (LR) library. Four of these cDNAs correspond to mRNAs that are positively or negatively regulated upon activation of photosynthesis. One clone represents a mRNA that accumulates transitorily at both transitions. Finally, LI818 cDNA identifies a new chlorophyll a/b-binding (cab) gene family whose mRNA accumulation is controlled by light and a circadian oscillator. The endogenous timing system controls LI818 mRNA accumulation so that it precedes the onset of illumination by a few hours. On the other hand, light affects LI818 mRNA levels independently of active photosynthesis.     

Characterization of lipopolysaccharide-induced macrophage gene expression

A cDNA library from LPS-treated murine peritoneal macrophages has been screened by differential hybridization with radiolabeled cDNA from untreated and LPS-treated macrophages. Six clones hybridizing with mRNA sequences present in LPS-treated cells but not in controls were selected for further characterization. When the recombinant bacteriophage DNA from each clone was used as a probe in Northern analysis of total RNA from LPS-treated macrophages, inducible mRNA ranging from 1.45 to 6.4 kb were seen. In five of six cases, the mRNA expression was undetectable in untreated macrophage cultures. All but one clone identified mRNA that were inducible even in the presence of cycloheximide, indicating the independence of such gene expression from protein synthesis; none of the genes were superinduced by this treatment. The time course of expression differed among the individual genes. Four were induced transiently, whereas two showed stable increasing accumulation through an 8-h period after stimulation. In addition, four of the genes were seen within 30 min of stimulation, whereas two were seen only after 2 to 4 h. Two genes were induced only by treatment with LPS, whereas four were also induced in response to other agents, including IFN-gamma, macrophage CSF, and PMA. The insert sequences from these recombinant clones did not hybridize with a set of cDNA encoding other inducible gene products, including TNF, IL-1, ornithine decarboxylase, c-myc, c-fos, JE, or KC. Thus, these six cDNA appear to encode inducible macrophage genes that are distinct from one another as well as from a selection of previously described early genes. Although their functional identity remains indeterminate, they may encode previously described early proteins induced in macrophages treated with LPS.     

Molecular cloning and characterization of cDNA for androgen-repressible rat liver protein, SMP-2

SMP-2 is a rat liver protein whose synthesis is influenced by both androgens and aging. The steady-state level of its mRNA is repressed by the androgen. Compared to the adult male, SMP-2 mRNA is found in higher amounts in the prepubertal and senescent male rat livers which show relative androgen insensitivity. A cDNA library in the plasmid pBR322 was constructed from the female rat liver which contains a high level of SMP-2 mRNAs. Recombinant plasmids were screened by differential colony hybridization to 32P-labeled single-stranded cDNAs from adult female and adult male hepatic poly(A)+ RNAs. From a total of 3500 recombinant clones, 11 highly female specific clones were identified. From these female specific colonies the SMP-2 cDNA-containing plasmid (pSP11) was identified by its ability to select an mRNA species whose translation product is immunochemically and electrophoretically indistinguishable from SMP-2. This insert represents a 571-base pair portion of the SMP-2 cDNA. Rescreening of the library at a high colony density using the 32P-labeled cDNA insert of pSP11 identified several positive clones with larger inserts. Hybrid-selected mRNA translation again confirmed these clones to carry SMP-2 cDNA sequences. The plasmid pSP4a containing a 1040-base pair cDNA insert of SMP-2 was characterized by DNA sequence analysis. The size of the cDNA insert of pSP4a is close to the estimated size of the SMP-2 mRNA. The cDNA sequence provides an open reading frame of 282 amino acid residues. A comparison of the translated amino acid sequence with the protein sequences of NBRF-PIR, PSQNEW, and LOSALA data bases did not establish any sequence homology with known proteins. Northern blot analysis using the 32P-labeled cDNA insert of pSP4a confirms the androgenic repression of the SMP-2 mRNA.     

Identification of iron responsive genes by screening cDNA libraries from suppression subtractive hybridization with antisense probes from three iron conditions

The goal of the present study is to identify genes that respond to iron availability. Suppression subtraction hybridization (SSH) was used to generate cDNA libraries from iron loaded and control human astrocytoma cells (SW1088). The cDNA libraries were screened with antisense cDNA probes obtained from mRNA isolated from astrocytoma cells exposed to three conditions: (i) normal media (control), (ii) deferoxamine treated (iron deficient) or (iii) iron loaded. The screening of the cDNA libraries with antisense probes from the three conditions enhanced the screening efficiency and decreased the number of false positives. Positive clones were identified and sequenced. The genes of interest were further analyzed by determining changes in hybridization signal on northern blots from astrocytoma cells exposed to iron or deferoxamine over different time intervals. Our analysis identified cDNAs corresponding to known iron responsive genes such as L-chain ferritin, but also revealed a number of mRNAs with novel sequences and mRNAs previously not known to be responsive to iron such as one of the ABC transporters and Thy-1 glycoprotein. Thus our results suggest that the expression of a number of genes may be influenced by changes in iron availability.     

Construction of two ordered cDNA libraries enriched in genes encoding plasmalemma and tonoplast proteins from a high-efficiency expression library

We constructed a high-efficiency expression library from Arabidopsis cDNA clones by introducing a poly (dC) stretch at the 5' end of the clones. This library enables the synthesis of proteins from all the cDNA clones present. We have screened the high-efficiency expression library with antibodies raised against total proteins from Arabidopsis plasmalemma and tonoplast. With the positive clones, we have constructed two cDNA ordered libraries enriched in genes encoding plasmalemma (522 clones) and tonoplast proteins (594 clones). Partial sequencing of both libraries shows that a high proportion (47%) of the clones encoded putative membrane proteins, or membrane-associated proteins. When sequenced, 55% of the cDNAs were new EST sequences for Arabidopsis, 26% were similar to genes present in other plants or organisms, and 29% were not referenced in any databank. Immunoscreening of the two cDNA ordered libraries with antibodies raised against proteins from Arabidopsis cells submitted to osmotic stress allows the selection of genes over- and under-expressed in stress conditions.     

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).