Expression In Eukaryotic Cells Research Articles

Poising chromatin for transcription

Interactions between cis-acting DNA sequences and trans-acting factors regulate gene expression in eukaryotic cells. In chromatin, these protein-DNA complexes frequently generate DNase I hypersensitive sites, are sometimes organized at or near the bases of chromosomal loops, and can instruct an entire linked domain to memorize the transcriptionally poised state.

Affinity chromatography of splicing complexes: U2, U5, and U4 + U6 small nuclear ribonucleoprotein particles in the spliceosome.

The splicing process, which removes intervening sequences from messenger RNA (mRNA) precursors is essential to gene expression in eukaryotic cells. This site-specific process requires precise sequence recognition at the boundaries of an intervening sequence, but the mechanism of this recognition is not understood. The splicing of mRNA precursors occurs in a multicomponent complex termed the spliceosome. Such an assembly of components is likely to play a key role in specifying those sequences to be spliced. In order to analyze spliceosome structure, a stringent approach was developed to obtain splicing complexes free of cellular contaminants. This approach is a form of affinity chromatography based on the high specificity of the biotin-streptavidin interaction. A minimum of three subunits: U2, U5, and U4 + U6 small nuclear ribonucleoprotein particles were identified in the 35S spliceosome structure, which also contains the bipartite RNA intermediate of splicing. A 25S presplicing complex contained only the U2 particle. The multiple subunit structure of the spliceosome has implications for the regulation of a splicing event and for its possible catalysis by ribozyme or ribozymes.

Sequence and expression of human estrogen receptor complementary DNA.

The mechanism by which the estrogen receptor and other steroid hormone receptors regulate gene expression in eukaryotic cells is not well understood. In this study, a complementary DNA clone containing the entire translated portion of the messenger RNA for the estrogen receptor from MCF-7 human breast cancer cells was sequenced and then expressed in Chinese hamster ovary (CHO-K1) cells to give a functional protein. An open reading frame of 1785 nucleotides in the complementary DNA corresponded to a polypeptide of 595 amino acids and a molecular weight of 66,200, which is in good agreement with published molecular weight values of 65,000 to 70,000 for the estrogen receptor. Homogenates of transformed Chinese hamster ovary cells containing a protein that bound [3H]estradiol and sedimented as a 4S complex in salt-containing sucrose gradients and as an 8 to 9S complex in the absence of salt. Interaction of this receptor-[3H]estradiol complex with a monoclonal antibody that is specific for primate ER confirms the identity of the expressed complementary DNA as human estrogen receptor. Amino acid sequence comparisons revealed significant regional homology among the human estrogen receptor, the human glucocorticoid receptor, and the putative v-erbA oncogene product. This suggests that steroid receptor genes and the avian erythroblastosis viral oncogene are derived from a common primordial gene. The homologous region, which is rich in cysteine, lysine, and arginine, may represent the DNA-binding domain of these proteins.

The domain structure and the function of poly(ADP-ribose) synthetase

Poly(ADP-ribose) synthetase is a chromatin-bound enzyme which synthesizes a protein-bound homopolymer of ADP-ribose utilizing NAD as a substrate. The characteristic nature of this enzyme is that it requires DNA for catalytic activity. The enzyme is rich in malignant tumor cells as well as in normal tissues where cell proliferation is very rapid. The enzyme has been purified to homogeneity from calf thymus, mouse testis and human placenta. The amino acid composition of these enzymes is very similar and a monoclonal antibody as well as antisera against the calf enzyme cross-reacts with mouse, chicken and human enzymes, suggesting that the antigenic structures of poly(ADP-ribose) synthetase are highly conserved in various animal cells. The native enzyme (Mr = 120K) is cleaved by limited proteolytic digestion into three different domains (Mr = 46K, 22K, 54K), the first containing the site for DNA binding, the second containing the site for automodification and the third containing the site for NAD binding. The DNA binding domain (Mr = 46K), like the native enzyme, has the ability to preferentially suppress nick induced random transcription initiation in a HeLa cell lysate, resulting in the production of run-off RNA initiated from the correct late promoter site on truncated DNA of adenovirus 2. The native enzyme poly(ADP-ribosyl)ates RNA polymerase and some other nuclear enzymes. These results, taken together, indicate that poly(ADP-ribose) synthetase plays a critical role in regulating gene expression in various eukaryotic cells.

Repression of simian virus 40 early transcription by viral DNA replication in human 293 cells.

The small DNA tumour virus simian virus 40 (SV40) has served as an excellent model for many studies on the mechanism and control of gene expression in eukaryotic cells. The SV40 early region produces two protein products. One product (large-T antigen) is known both to repress early viral transcription and to stimulate viral replication by binding to specific sites in the origin-promoter region. The early promoter has several similarities to other RNA polymerase II promoters, for example, it possesses a TATA box, an upstream element and an enhancer. However, the SV40 early promoter differs from other known RNA polymerase II promoters in that the origin of viral DNA replication is embedded within it. Here we show that the SV40 early region is expressed at an extremely low level following its introduction ito human 293 cells, contrasting with results observed in a large number of other cells lines. We show further that the lack of expression is due to repression of transcription from the SV40 early promoter by viral DNA replication which occurs efficiently in 293 cells.

Growth hormone gene expression in eukaryotic cells directed by the Rous sarcoma virus long terminal repeat or cytomegalovirus immediate-early promoter

The cytomegalovirus (CMV) immediate-early (IE) gene-regulatory region was found to be three- to fourfold more efficient than the Rous sarcoma retroviral long terminal repeat (LTR) in promoting expression of the bovine growth hormone (bGH) gene by rat GH 3 cells.

Expression of cloned genes microinjected into cultured mouse and human cells

The direct approach of capillary microinjection was used to study exogenous gene expression in mouse and human cells. The efficiencies of biochemical and morphological transformation in mouse cells and the differential ras gene expression in mouse and human cells were determined. Biochemical transformation efficiency of mouse cells after microinjection was ≈2.5% with the thymidine-kinase plasmid and ≈30% with the neomycin-resistant gene, pSV2 neo, which contains the SV-40 enhancer sequence. Mouse NIH3T3 cells microinjected with genes of the ras family—including viral isolates, the human bladder oncogene EJ, and N- ras—produced morphologically transformed foci with an efficiency of 10–20%, whereas the normal human cellular homologue of the ras gene in Harvey sarcoma virus showed an efficiency of 0.5–1%. Expression of v- ras genes was compared in mouse and human cells by both immunofluorescence detection of the ras protein, p21, 24 hr after microinjection and by the focus assay. The data suggest that microinjection is a useful approach for the investigation of early exogenous gene expression and control in eukaryotic cells and has the potential to allow insight into oncogenic events.

The presence of cysteine in the cytoplasmic domain of the vesicular stomatitis virus glycoprotein is required for palmitate addition.

The transmembrane glycoprotein (G protein) of vesicular stomatitis virus (VSV) is known to contain 1-2 mol of covalently linked fatty acid (palmitate) per mol of protein. G protein is oriented in cellular membranes such that the carboxyl-terminal 29 amino acids protrude into the cytoplasm. We have obtained expression in eukaryotic cells of mutagenized cDNA clones that encode VSV G proteins lacking portions of this cytoplasmic domain. Labeling of these truncated proteins with [3H]palmitate indicated that the palmitate might be linked to an amino acid residue within the first 14 residues on the carboxyl-terminal side of the transmembrane domain. Using oligonucleotide directed mutagenesis, we changed the single codon specifying cysteine in this domain to a codon specifying serine. Expression of this mutant gene results in synthesis of a G protein lacking palmitate. We suggest that linkage of palmitate to G protein is through the cysteine in the cytoplasmic domain and that such a linkage may occur in many viral and cellular glycoproteins. The G protein lacking palmitate is glycosylated and is transported normally to the cell surface.

Exposure of JB-6 mouse epidermal cells to 12-O-tetradecanoyl-phorbol-13-acetate is not accompanied by a significant change in total DNA-cytosine methylation.

The extent of methylation of the cytosine bases in DNA is believed to be a major factor influencing gene expression in eukaryotic cells. We have asked whether the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) alters the amount of 5-methylcytosine in DNA. The amount and relative distribution of 5-methylcytosine in the DNA of two subclones of the JB-6 mouse epidermal cell line were determined respectively by high performance liquid chromatography and digestion with the restriction enzymes MspI and HpaII. Exposure to TPA for up to several cell generations had no detectable effect on the degree of DNA methylation (3.9% of the total cytosine) in the two JB-6 lines or Friend erythroleukemia cells. Reduced methylation was readily detected in DNA extracted from cells exposed to 5-azacytidine. The data suggest that tumor promotion (at least that induced by TPA) is likely not the consequence of a generalized elevation or reduction in the amount of 5-methyl-cytosine in the DNA.

Molecular cloning of human immune interferon cDNA and its expression in eukaryotic cells.

Starting with mRNA derived from Staphylococcal enterotoxin A induced human splenocytes, dsDNA was synthesized and inserted into unique BamHI site of the eukaryotic expression vector pSV529 (1). A recombinant plasmid containing human immune interferon (IFN-gamma) cDNA was identified by hybridization of plasmid inserted DNA bound onto nitrocellulose filters with mRNA derived from SEA-induced splenocytes, translation of the eluted RNA in Xenopus laevis oocytes and assaying for IFN activity. Plasmids containing the entire human IFN-gamma cDNA sequence were identified by colony hybridization and were sequenced. A unique coding region was identified which predicted a protein of 166 amino acids, the 20 N-terminal amino acids of which presumably represent a signal peptide. After transfection of monkey cells with plasmid DNA isolated from one of the recombinant clones (pHIIF-SV-gamma 1), IFN was excreted into the culture medium. This IFN was not distinguishable from human IFN-gamma by serological criteria or by cell target species specificity.

Short-lived cytoplasmic regulators of gene expression in cell cybrids.

Somatic cell hybridization is a valuable tool for investigation the control of gene expression in eukaryotic cells. Studies of hybrid cells, heterokaryons, reconstructed cells and cybrids (cytoplasmic hybrids) have suggested that cytoplasmic factors may be involved in this regulatory process. Unfortunately, studies of this kind usually require that hybrid or modified cells be maintained for some time in a selective environment during which chromosomal losses or other changes may modify the genetic functions of the cells and thus vitiate conclusions about the mechanism of gene regulation. We report here the preparation of cybrids between enucleated mouse fibroblasts (Cl-1-D) and differentiated rat hepatoma cells (Fao) and the use of a combination of histological techniques to identify these modified cells early after fusion without the use of selective media. We found that albumin production in most cybrids was suppressed (extinguished) at 12-20 h after fusion but was restored by 48 h. These results suggest that there is a cytoplasmic factor in the fibroblast which exerts negative control over expression of the albumin gene, but which in the absence of the fibroblast nucleus, is not renewed and therefore short-lived.

Role of Chromatin Structure, Histone Acetylation, and the Primary Sequence of DNA in the Expression of SV40 and Polyoma in Normal or Teratocarcinoma Cells

Publisher Summary The extensive knowledge about the viral genome and its minichromosomes permits a more detailed analysis of the nature of the regulating elements governing the expression of the viral genome at the level of the DNA primary sequence and at the level of the chromatin. The region containing the initiation sites for late, and probably for early, transcription has a unique conformation in the viral minichromosomes. This region is devoid of nucleosomes in 5% to 20% of the viral chromosomes, as detected by the electron microscopy and by DNase I digestion of nuclei isolated from infected cells. The chapter discusses on analyzing the SV40-transformed rat cells in an attempt to probe the structure of the integrated viral genome and its regulatory regions in these cells. It is clear, from both sequence comparison and transcription studies in vitro, that specific sequences are responsible for “promoter” recognition by eukaryotic RNA polymerases and their cofactors. The sequence studies of polyoma mutants that grow on embryonal carcinoma cells suggest that the promoters recognized in a differentiated cell may differ from those recognized by an embryonic cell. This may be analogous to the modifications of the transcription machinery at both the enzyme and DNA levels after phage SPOl infection of Bacillus subtilis. Embryonic development and differentiation may thus be accompanied by the changes in RNA polymerase II or its cofactors and by the changes in the sequence of the initiation sites recognized on the cellular genome. The control of gene expression by both the primary DNA sequence and the structure of chromatin may permit more refined and tighter control of gene expression in eukaryotic cells.

Electrophoretic profiles of nonhistone nuclear proteins of human hearts with muscular subaortic stenosis.

Muscular subaortic stenosis (MSS) is a genetically determined cardiomyopathy, whereas right ventricular infundibular hypertrophy (IH) apparently is an acquired condition. Since genetic expression in eukaryotic cells may be regulated primarily by DNA-associated proteins, we isolated and characterized the proteins of heart nuclei from nine patients with MSS, eight with IH, and two with normal (N) hearts. More than 150 proteins could be identified by two-dimensional polyacrylamide gel electrophoresis. Proteins in the entire region from pH 7.0 to 9.0 with molecular weights (Mr) ranging from 35,000 to 41,000 and a protein focusing from pH 5.2 to 5.3 with Mr of 55,000 were strikingly reduced in MSS. Again the electrophoretic patterns of N and IH were similar. The electrophoretic patterns of nonhistone nuclear protein (NHNP) in MSS relative to N showed a striking resemblance to those demonstrated previously for the early stage (myolytic phase) of hamster cardiomyopathy relative to the matched control. Since NHNP interacting with DNA appears to play a major role in genetic expression, it is possible that some of the manifestations of MSS could be due to different components of NHNP in the affected hearts.

Splicing of SV40 mRNAs: A Novel Mechanism for the Regulation of Gene Expression in Animal Cells

Clarification of the complicated steps involved in transcription and processing of mRNA seems essential in order to understand the controls involved in gene expression in eukaryotic cells.

Drosophila Gene-Enzyme Systems

The genetic analysis of morphological mutants of drosophila in the past years has led to a nearly complete catalog of specific genetic sites that control many features of the adult phenotype. An approach to understanding drosophila gene action has relied on studying mutations of genes for specific enzymes that affect subtle secondary characteristics of the enzyme, have little or no effect on enzymatic properties in vivo , and do not impair the viability of the mutant organism. Several different gene-enzyme systems in drosophila have been described and genetically characterized, thereby adding substantially to the understanding of genetic expression in eukaryotic cells. The chapter focuses on the specific properties of individual gene-enzyme systems that have been amenable to analysis and hold promise for additional investigation, and discusses the genetic nomenclature, specific gene-enzyme systems, dosage compensation of gene activity, genetic organization, genetic regulation of enzyme formation, and such. Emphasis on enzyme polymorphisms is given with the view that the results of these comparisons can often provide information on possible interactions of gene systems and products.

Regulation of Gene Expression in Eukaryotic Cells.

Regulation of Gene Expression in Eukaryotic Cells.

Binding of chromosomal non histone proteins to DNA and to nucleohistones. Effect of in vitro phosphorylation

1. Introduction The possibility that chromosomal non-histone pro- teins (NHP) could be regulators of gene expression in eukaryotic cells is suggested by chromatin reconstitu- tion experiments and by DNA/RNA hybridation ana- lyses [l-2]. These experiments showed that NHP modi- fied transcription in a manner characteristic of the tissue of origin. But the mechanisms by which the NHP was able to interact and to modify the transcription are not completely clear. However, several studies have implied that NHP is able to stimulate the tem- plate activity of nucleohistones, at least partly, through the presence of phosphoproteins and of protein kinases [3]. Similarly, NHP modifications are associated with the stage of the cell cycle and with the differentiation of cells during development and aging [4-81. A direct role for NHP cannot be excluded since some NHP does bind to DNA [9,10] . In chromatin however, large parts of DNA are covered with histones [ 11 ,121. Histones have been demonstrated to play a role in the structure of DNA and in the chromatin conformation [13,14]. They have an inhibitory effect on the transcription availability of DNA. This inhibi- tory action can be partially suppressed by histone- phosphorylation [ 151. In this work we have studied comparatively the binding of NHP to DNA and to formaldehyde-treated nucleohistones, this last technique allowing a cova- lent binding of histones to DNA [12,16]. DNA and formaldehyde-treated nucleohistones (FNH) were f=ed on a cellulose matrix and poured into a column. A small NHP fraction remained bound and could be

The regulation of gene expression in eukaryotic cells

A new hypothesis for the regulation of gene expression in eukaryotic cells is proposed. This hypothesis suggests that small segments of activator RNA select sites for gene transcription by base pairing with unique base sequences at the 3′ end of DNA gene sites and act as primer for RNA transcription of these genes. Unlike previous theories that have been proposed, this theory postulates a source for this activator RNA, as breakdown products of high molecular weight RNA and offers an explanation for the programmed schedule in the pattern of gene transcription during development as well as the generally stable phenotypic characteristics of cells.

Discontinuous Antibody Secretion during the Secondary Response to Sheep Erythrocytes in Vitro

Abstract Antibody-producing lymphocytes provide a unique model for studying cellular differentiation and patterns of gene expression in eukaryotic cells as recently reviewed by Lerner and Dixon (1). The relationship between gene expression for antibody synthesis and the cell cycle has been studied in several established antibody-producing cell lines with varying results. Human lymphoid cell lines consistently show the highest level of antibody synthesis in the late G1, early S (2–5), and possibly G2 (5) stages of the cell cycle and a reduced synthesis around metaphase. Similarly, Byars and Kidson (6) demonstrated that established mouse myeloma cells also exhibit maximal antibody synthesis in late G1 and early S, and Gandini-Attardi et al. (7) suggest that mouse myeloma cells contain more antibody in S than in other stages of the cycle. On the other hand, studies by Cowan and Milstein (8) and Liberti and Baglioni (9), also using mouse myeloma cell lines, indicate that antibody is synthesized at a fairly constant rate throughout the cell cycle.

Nonhistone chromosomal proteins and gene regulation.

Evidence from several model systems suggests that nonhistone chromosomal proteins may regulate gene expression in eukaryotic cells. The data indicate that the synthesis of new species of nonhistone chromosomal proteins as well as modifications of preexisting nonhistone chromosomal proteins are involved in the control of transcription. However, from the vast number of proteins included in this class, it is apparent that, in addition to regulating the transcription of defined genome loci, the nonhistone chromosomal proteins include enzymes that have a general function, proteins that are involved in determining the structure of chromatin, as well as proteins that serve as recognition sites for binding of regulatory macromolecules. The presence of a nucleoplasmic pool of nonhistone chromosomal proteins which may exchange with the chromatin has also been reported (89). While it is clear that the nonhistone chromosomal proteins play a key role in the regulation of gene expression, the exact manner in which they interact with the genome to initiate, modify, or augment the transcription of specific RNA molecules remains to be resolved.