Cyclin mRNA and protein expression in recombinant interleukin 2-stimulated cloned murine T lymphocytes

Expression of cyclin, a non-histone nuclear protein, during recombinant interleukin 2 (rIL2)-driven cell-cycle progression of cloned T lymphocytes has been assessed. We found that expression of cyclin protein, as detected by immunofluorescence, is tightly associated with proliferation, and not merely S-phase, of L2 cells stimulated with rIL2. Cyclin immunofluorescence was detected in all cell-cycle phases (G1/S/G2/M, as detected by flow cytometry) of proliferating L2 cells. Accumulation of cyclin mRNA levels was induced as early as 1 h after stimulation, was maximal at 25-49 h, and remained elevated throughout stimulation, as detected by Northern blot analysis. A cDNA-encoding murine cyclin was cloned from a cDNA library prepared from IL2-stimulated cloned T cells. The sequence of the 5' end of the murine cyclin cDNA was determined and found to be 88% and 82% similar to the sequences of cDNA clones encoding rat and human cyclin, respectively. The present studies demonstrate that cyclin protein and mRNA accumulation are highly regulated during IL2-induced proliferation of a cloned T cell. These data provide a framework for addressing the molecular mechanisms regulating cyclin gene expression during cellular proliferation.     

Differential screening of a cDNA library with cDNA probes amplified in a heterologous host: isolation of murine GRP78 (BiP) and other serum-regulated low-abundance mRNAs

A novel method was used to screen differentially a cDNA library for clones representing serum-regulated mRNA species of low abundance. To increase the amount of probe available for screening, the cDNA probe was cloned and amplified. Two separate cDNA 'probe' libraries were constructed in the Escherichia coli plasmid vector pDE613, using poly(A)+mRNA from murine cells at 0 and 16 h after stimulation of a G0 population. Radiolabelled plasmid DNA from each library was hybridized sequentially to colony blots of the third 'target' library, constructed with mRNA from serum-stimulated cells in the Bacillus subtilis vector pBD214. Differential screening of the target cDNA library with the two probe libraries identified novel murine cDNA clones, some representing cytoplasmic poly(A)+RNA species of low (0.01%) abundance, accumulating after serum stimulation of a quiescent mouse embryo fibroblast population. One cDNA clone was found to correspond to mitochondrial 16S rRNA and a second was identified as the murine equivalent of previously described cDNA clones for the hamster 78-kDa glucose-regulated protein (GRP78) and the rat immunoglobulin heavy-chain-binding protein. GRP78 mRNA has not previously been recognized as a serum-inducible message.     

Differential expression of metastasis-associated cell surface glycoproteins and mRNA in a murine large cell lymphoma

A metastatic variant cell subline of the Abelson virus-transformed murine large lymphoma/lymphosarcoma RAW117 has been selected in vivo ten times for liver colonization. Highly metastatic subline RAW117-H10 forms greater than 200 times as many gross surface liver tumor nodules as the parental line RAW117-P. Analysis of cellular proteins and glycoproteins indicates reduced expression of murine Moloney leukemia virus-associated p15, p30, and gp70, and increased expression of a sialoglycoprotein, gp150, in the highly metastatic H10 cells. Northern analyses of oncogene expression suggested that mRNA of various oncogenes was expressed equally or not expressed in the RAW117 cells of differing metastatic potential. Differential gene expression was examined using a cDNA library of 17,600 clones established from poly A+ mRNA isolated from H10 cells. The cDNA library was screened by the colony hybridization technique using probes made from both RAW117-P and -H10 cells. Approximately 99.5% of these cDNA clones were expressed identically in P and H10 cells. Of the few differentially expressed cDNA clones (approx. 150/17,600), one-half of these were identified as Moloney leukemia virus sequences in a separate probing with a radiolabeled Moloney leukemia virus probe. The remainder of the differentially expressed mRNA detected by colony hybridization of the cDNA library were expressed at higher levels (approx. 1/6) or lower levels (approx. 1/3) in the highly metastatic H10 cells.     

Cloning and expression of murine lymphotoxin cDNA

The murine lymphotoxin (LT) gene has been cloned and used to identify cDNA clones in a library prepared from activated murine T cell mRNA. A recombinant murine genomic library was screened with a human lymphotoxin cDNA probe, resulting in the isolation of the entire LT gene. The murine LT gene structure is similar to the human gene, containing three intervening sequences. An activated murine T cell cDNA library was prepared with poly(A)+ RNA isolated 7 hr after concanavalin A stimulation of an L3T4+ interleukin 2-dependent murine T cell clone. Two colonies of the cDNA library that contained inserts that hybridized with the murine LT gene probe were sequenced and were used to construct expression plasmids. The amino acid sequence deduced from the cDNA indicates that murine LT is highly homologous to human LT (74%) and is related to murine tumor necrosis factor (35% homology). The cDNA was transcribed and was translated in vitro, and was expressed in COS-1 cells. This has resulted in the production of LT biological activity.     

Cloning of alpha 2u globulin cDNA using a high efficiency technique for the cloning of trace messenger RNAs

An extremely high-efficiency technique is described for cloning double-stranded (ds) cDNAs in Escherichia coli. The method, which uses two synthetic oligonucleotide linkers rather than one, results in approx. 200--500 recombinant clones per ng of ds cDNA. This technique was used to clone a cDNA comprising 95% of the full length of the mRNA of alpha 2u globulin, a male rat liver protein, which represents approx. 1% of hepatic messenger RNA. The cloned probe was applied to study the complex hormone controls of alpha 2u globulin mRNA in male and female rats.     

Vector-capping: a simple method for preparing a high-quality full-length cDNA library.

Full-length cDNAs play an essential role in identifying genes and determining their promoter regions. Here we describe a simple method for constructing a full-length cDNA library, which has the following advantages: (i) it consists of only three steps including direct ligation between a vector and a cDNA strand using T4 RNA ligase, (ii) it contains neither a PCR process generating mutations nor restriction enzyme treatment causing truncation of cDNA, (iii) the intactness of cDNA is assured due to the presence of an additional dGMP at its 5' end, (iv) approximately 95% of cDNA clones are full-length when cultured cells or fresh tissues are used, (v) several micrograms of total RNA without mRNA purification is sufficient for preparation of a library containing >10(5) independent clones, and (vi) a long-sized full-length cDNA up to 9.5 kbp can be cloned. This method will accelerate comprehensive gene analysis in a variety of eukaryotes.     

Cloning of mRNA nucleotide sequences amplified during DNA replication in the regenerating liver

A library of double-stranded cDNA has been constructed using the mRNA of regenerating rat liver 20 hr after partial hepatectomy. The differential screening of the library with the regenerating liver specific and the resting-liver-specific single-stranded cDNA probes has identified 11 cDNA clones which sequences are preferentially expressed in regenerating rat liver. The RNA dot blot hybridization has shown that levels of RNA complementary to these clones are 3 to 8-fold higher in dividing cells as compared with resting cells.     

Characterization of two mRNA species encoding human estradiol 17 beta-dehydrogenase and assignment of the gene to chromosome 17

Using two 33-mer synthetic oligonucleotides derived from the amino acid sequence of the catalytic site of estradiol 17 beta-dehydrogenase (E2DH) and polyclonal antibodies raised against the enzyme purified from human placenta, clones were isolated from a lambda gt11 human placental cDNA library. A 327-amino acid sequence was deduced from cDNA sequencing. Two mRNA species have been identified in poly(A)+ RNA from human placenta, a major species migrating at 1.3 kb while a minor one is found at approx. 2.2 kb. Primer extension and S1 nuclease analysis indicate that the major mRNA species starts 9-10 nucleotides while the minor mRNA starts 971 nucleotides upstream from the ATG initiating codon, respectively. Sequence analysis of the longest cDNA clone (2092 bp) shows that it possesses identical coding and non-coding sequences in the regions of overlap with the shorter cDNA clones. The 32P-labeled 5' non-coding fragment hybridizes only to the 2.2 kb band, thus providing evidence for the existence of two distinct mRNA species which differ in their 5' noncoding regions. Using hp E2DH-36 cDNA as a probe for in situ hybridization of translocated chromosomes, the human E2DH gene was localized to the q11-q12 region of chromosome 17.     

Isolation and characteristics of clones complementary to mRNA of the murine cellular tumor antigen p53

43 cDNA clones specific for murine cellular tumor antigen p53 were isolated from a library constructed using 17S fraction of mRNA from the SV40 transformed murine fibroblasts (SVT2). These clones contain the whole coding region of p53 mRNA and the most of non-translated sequences. A plasmid containing 1.8 kb insert of p53 cDNA was constructed. The p53 specific insert in this plasmid was colinear with p53 mRNA, as revealed by S1 nuclease analysis. 5'-region of the p53 gene comprising non-translated and promoter areas was cloned from the mouse genomic library. A combined clone containing promoter and the whole region, corresponding to p53 mRNA has been constructed.     

Cloning of the cDNA encoding phenylalanyl tRNA synthetase regulatory alpha-subunit-like protein whose expression is down-regulated during differentiation.

Hybrid polar compounds (HPCs), such as suberoylanilide hydroxamic acid (SAHA), induce differentiation of transformed cells. Differential display of RNA was used to identify genes whose expression is changed during SAHA-induced differentiation of murine erythroleukemia (MEL) cells. One such cDNA was identified whose mRNA level decreased by 50% after 8h of SAHA treatment as determined by Northern blot analysis. The full-length cDNA (1944bp in length) was cloned by sequencing of an EST clone and rapid amplification of 5' cDNA ends (5'-RACE). The predicted amino acid sequence is 589 amino acids and shares 45% identity with the yeast cytoplasmic phenylalanyl tRNA synthetase (PheRS) regulatory alpha-subunit. Human EST clones which share over 90% identity of predicted amino acid sequence with this cDNA map to chromosome 2 near the paired box homeotic gene 3 (PAX3) locus, a region syngenic to mouse chromosome 1. This is the first report of the cloning of the full-length cDNA for the murine PheRS regulatory alpha-subunit-like protein. The level of PheRS alpha-subunit-like mRNA is regulated during differentiation but not during cell cycle progression.     

Cells transformed by a wide variety of agents express higher abundance levels of some cellular RNA species

A cDNA-cloned library was prepared from mRNA synthesized by SV40-transformed mouse cells. Eleven cDNA clones were selected based on their ability to hybridize higher levels of mRNA in SV40-transformed 3T3 cells than in 3T3 cells. These cDNA clones were employed to screen the steady-state levels of cytoplasmic RNAs in a wide variety of viral (SV40, polyoma, adenovirus, and Rous sarcoma virus) and nonviral (methylcholanthrene, embryonal carcinoma) transformed cell lines. Two of the cDNA clones—A17 and 104—detected greater than 40–100-fold higher levels of mRNA in all the transformed cell lines tested when compared to nontransformed cells (3T3, C3HEF). The levels of mRNA complementary to these two cDNAs were regulated in a temperature-sensitive fashion (87–100-fold) in both SV40tsA- and RSV ts-src-transformed murine cell lines. These two cDNA clones detected greater than 100-fold, higher levels of complementary RNA derived from SV40 tumor tissue than in normal mouse liver. RNA species complementary to cDNA clones A17 or 104 were not detected in either actively growing nontransformed cells or in serum-stimulated 3T3 cells. The abundance levels of mRNAs detected by these two cDNA clones appear to be regulated 100-fold or greater by the transformed state, independent of the transforming agent. The higher levels of these RNA species detected in transformed mouse cells appear not to be solely regulated by the state of growth of nontransformed cells.     

cDNA cloning and characterization of interleukin 2-induced genes in a cloned T helper lymphocyte

Clonal expansion of antigen-specific lymphocytes is an important aspect of the immune response. Interleukin 2 (IL2) is largely responsible for the amplification of antigen-specific T cells. In this study, the changes in gene expression accompanying interleukin 2 stimulation of T cells are examined, using a cloned T helper lymphocyte line as a model system. To isolate cDNA clones of IL2-induced genes, a cDNA library was screened by differential hybridization. Twenty-one different cDNA clones were isolated by this method, comprising six glycolytic enzymes, vimentin, alpha-tubulin, beta-actin, gamma-actin, ERp99, elongation factor 2, ribosomal phosphoprotein P1, the DNA-binding protein dbpB/YB-1, as well as seven clones which do not correspond to any previously described sequences. These clones are used to study the time course of expression and the sensitivity to cycloheximide inhibition of IL2-induced mRNAs. In addition, the tissue specificity of the unidentified mRNAs is examined, and two of these are shown to be expressed at high levels in normal mouse brain, with much lower or undetectable levels in the other tissues tested. These cDNA clones will be useful in future studies to determine the molecular basis of IL2-induced gene expression.     

Brain-specific expression of MAP2 detected using a cloned cDNA probe

We describe the isolation of a set of overlapping cDNAs encoding mouse microtubule associated protein 2 (MAP2), using an anti-MAP antiserum to screen a mouse brain cDNA expression library cloned in bacteriophage lambda gt11. The authenticity of these clones was established by the following criteria: (a) three non-identical clones each expressing a MAP2 immunoreactive fusion protein were independently isolated from the expression library; each of these clones cross-hybridized at the nucleic acid level; (b) anti-MAP antiserum was affinity purified using nitrocellulose-bound fusion protein; these antibodies detected only MAP2 in an immunoblot experiment of whole brain microtubule protein; (c) a series of cDNA "walking" experiments was done so as to obtain a non-overlapping cloned fragment corresponding to a different part of the same mRNA molecule. Upon subcloning this non-overlapping fragment into plasmid expression vectors, a fusion protein was synthesized that was immunoreactive with an anti-MAP2 specific antiserum. Thus, a single contiguous cloned mRNA molecule encodes at least two MAP2-specific epitopes; (d) the cloned cDNA probes detect an mRNA species in mouse brain that is of a size (approximately 9 kb) consistent with the coding capacity required by a 250,000-D protein. The MAP2-specific cloned cDNA probes were used in RNA blot transfer experiments to assay for the presence of MAP2 mRNA in a variety of mouse tissues. Though brain contained abundant quantities of MAP2 mRNA, no corresponding sequences were detectable in RNA prepared from liver, kidney, spleen, stomach, or thymus. We conclude that the expression of MAP2 is brain-specific. Use of the MAP2 specific cDNA probes in genomic Southern blot transfer experiments showed the presence of a single gene encoding MAP2 in mouse. The microheterogeneity of MAP2 is therefore ascribable either to alternative splicing within a single gene, or to posttranslational modification(s), or both. Under conditions of low stringency, the mouse MAP2 cDNA probe cross-hybridizes with genomic sequences from rat, human, and (weakly) chicken, but not with sequences in frog, Drosophila, or sea urchin DNA. Thus, there is significant interspecies divergence of MAP2 sequences. The implications of the above observations are discussed in relationship to the potential biological function of MAP2.