Showing papers on "Gene expression published in 1983"

Cloning and expression of human tissue-type plasminogen activator cDNA in E. coli

Abstract: Bacterial clones containing human tissue-type plasminogen activator (t-PA) cDNA sequences were identified in a cDNA library prepared using gel-fractionated mRNA from human melanoma cells. A plasmid containing the Escherichia coli trp promoter and the cDNA sequence coding f or the 527-amino acid mature t-PA protein was constructed for expression in E. coli. A polypeptide was produced having the fibrinolytic properties characteristic of authentic human t-PA.

Amplification and expression of the c- myc oncogene in human lung cancer cell lines

Abstract: Genetic changes involving the c-myc oncogene have been observed in human tumours. In particular, the c-myc gene is translocated in Burkitt's lymphoma and is amplified in the human promyelocytic leukaemia cell line, HL-60, which contains double minute chromosomes (DMs). More recently, an amplified c-myc gene has been positioned on a chromosomal homogeneous staining region (HSR) in a human colon cancer cell line, COLO 320, with neuroendocrine properties. Furthermore, c-myc is expressed in increased amounts in some human tumour lines, and in some cases, human small cell lung cancers (SCLC) contain DMs and HSRs. These findings prompted us to study the c-myc gene and its RNA expression in a series of human lung cancer cell lines. We now report amplification and expression of the c-myc oncogene in a system other than B-cell lymphomas, namely human lung cancer. Of 18 human lung cancer cell lines tested, 8 showed an amplified 12.5-kilobase (kb) EcoRI c-myc DNA band. Of particular interest are five SCLC lines with a high degree of c-myc DNA amplification (20-76-fold) and greatly increased levels of c-myc RNA. All five lines reside in the variant class of SCLC (SCLC-V) characterized by altered morphology, lack of expression of some SCLC-differentiated functions and more malignant behaviour than pure SCLC. Three of the five lines which have been karyotyped also contain DMs or HSRs. The finding of a greatly amplified c-myc gene in all cell lines of the SCLC-V class examined strongly suggests a role for the c-myc gene in the phenotypic conversion and malignant behaviour of human lung cancer.

Metallothionein-human GH fusion genes stimulate growth of mice

Abstract: The promoter or regulatory region of the mouse gene for metallothionein-I was fused to the structural gene coding for human growth hormone. These fusion genes were introduced into mice by microinjection of fertilized eggs. Twenty-three (70 percent) of the mice that stably incorporated the fusion genes showed high concentrations of human growth hormone in their serum and grew significantly larger than control mice. Synthesis of human growth hormone was induced further by cadmium or zinc, which normally induce metallothionein gene expression. Transgenic mice that expressed human growth hormone also showed increased concentrations of insulin-like growth factor I in their serum. Histology of their pituitaries suggests dysfunction of the cells that normally synthesize growth hormone. The fusion genes were expressed in all tissues examined, but the ratio of human growth hormone messenger RNA to endogenous metallothionein-I messenger RNA varied among different tissues and different animals, suggesting that expression of the foreign genes is influenced by site of integration and tissue environment.

Beta-galactosidase gene fusions for analyzing gene expression in Escherichia coli and yeast

Abstract: Publisher Summary Gene fusions can be constructed either in vivo , using spontaneous nonhomologous recombination or semi-site specific transposon recombination, or in vitro , with recombinant DNA technology. This chapter describes in vitro methods and lists some recently developed β -galactosidase gene fusion vectors. With these methods, gene-controlling elements from any source can be fused to the β -galactosidase structural gene and examined in the prokaryote bacterium Escherichia coli or the lower eukaryote yeast Saccharomyces cerevisiae. β -galactosidase gene fusions can be constructed both with transcription initiation control signals and with transcription plus translation initiation control signals. The β -galactosidase gene is convenient for making translational fusions because it is possible to remove its translation initiation region along with up to at least the first 27 amino acid codons without affecting β -galactosidase enzymic activity. The focus here is on these transcription-translation fusions because they provide all the gene initiation signals from the other gene. β -Galactosidase expression from a gene fusion can be used not only to measure gene expression and regulation, but also to isolate mutations and additional gene fusions.

Cell-specific expression controlled by the 5′-flanking region of insulin and chymotrypsin genes

Abstract: DNA sequences containing the 5′-flanking regions of the insulin and chymotrypsin genes were linked to the coding sequence of the chloramphenicol acetyltransferase (CAT) gene. The insulin gene recombinant elicits preferential expression of CAT activity when introduced into cells producing insulin; similarly, the chymotrypsin gene recombinant elicits preferential expression in chymotrypsin-producing cells. Sequences located upstream of previously defined transcriptional control elements are essential for efficient expression in both cases.

Expression and regulation of Escherichia coli lacZ gene fusions in mammalian cells.

Abstract: Gene fusions between the Escherichia coli lacZ gene and DNA segments containing the simian virus 40 early promoter or the mouse mammary tumor virus (MMTV) promoter direct the synthesis of functional beta-galactosidase in Cos 7 monkey cells and mouse Ltk-cells. Enzymatic activity was measured either 72 h after transfection or in stable transformants. The sensitive beta-galactosidase assay was used to measure gene expression and to optimize the efficiency of DNA-mediated transfection. Glucocorticoids stimulated the production of beta-galactosidase when lacZ was fused to the hormonally regulated MMTV promoter.

Actively transcribed genes are associated with the nuclear matrix

Abstract: In the chicken oviduct, it has been well documented that steroid hormones stimulate the transcription of specific genes such as the ovalbumin gene. In addition to the presence of specific hormone receptors in the tissue, gene expression seems to require that target genes exist in large DNase I sensitive chromosomal domains. This structure appears necessary but not sufficient for transcriptional activation. In search of still other levels of control, we have investigated the interactions of genes with the nuclear matrix, a structure which has been implicated in DNA synthesis, transcription and RNA processing. Here we have isolated nuclear matrix and used a nondegradative method to fractionate nuclear DNA based on its preferential association with the matrix. The preparation was digested with a restriction enzyme and both matrix-bound and released DNAs were recovered. We found that only actively expressed genes were associated with the matrix. Furthermore, within a 100-kilobase (kb) DNase I sensitive chromosomal domain, only the transcribed regions were associated with the matrix. This association was shown to be reversible when hormone was withdrawn. Our results suggest that the nuclear matrix is the site of nuclear transcription and may represent another potential level of control for regulation of gene expression in the eukaryotic cell.

Two distinct mechanisms regulate the levels of a cellular tumor antigen, p53.

Abstract: The steady-state levels of p53 protein and p53 mRNA in transformed and nontransformed cells were examined to elucidate the mechanisms controlling expression of p53. mRNA levels were determined by Northern blot hybridization analysis, employing a p53-specific cDNA clone (M. Oren and A.J. Levine, Proc. Natl. Acad. Sci. U.S.A. 80:56-59, 1983), and protein levels were determined by the Western blotting technique. Analysis of p53 mRNA revealed a single polyadenylated mRNA species migrating at ca. 18S. Levels of p53 mRNA in simian virus 40-transformed cell line (SVT2) and in an homologous nontransformed cell line (3T3) were equivalent, although the steady-state levels of p53 protein were 25- to 100-fold higher in the SVT2 cells than in the 3T3 cells. A study with a non-virus-transformed cell system revealed a different result. Embryonal carcinoma cells (F9) were found to have nearly 20-fold higher levels of p53 mRNA in comparison with differentiated benign progeny cells. In this system the difference in p53 mRNA levels corresponded to the difference in p53 protein levels. Pulse-chase experiments were performed to study the half-life of p53 protein in these four types of cells. The turnover of p53 protein occurred with biphasic kinetics. In addition, it was found that protein synthesis inhibitors placed in the medium during the chase period prevented the turnover of p53 protein in transformed cells, but not in nontransformed (3T3) cells. These results provide evidence that the regulation of p53 expression in cells can occur at the level of p53 mRNA abundancy or p53 protein stability depending upon the experimental system under study, and that a regulated degradation process controls the turnover of p53 protein.

Gene expression in rat brain

Abstract: 191 randomly selected cDNA clones prepared from rat brain cytoplasmic poly (A)+ RNA were screened by Northern blot hybridization to rat brain, liver and kidney RNA to determine the tissue distribution, abundance and size of the corresponding brain mRNA. 18% hybridized to mRNAs each present equally in the three tissues, 26% to mRNAs differentially expressed in the tissues, and 30% to mRNAs present only in the brain. An additional 26% of the clones failed to detect mRNA in the three tissues at an abundance level of about 0.01%, but did contain rat cDNA as demonstrated by Southern blotting; this class probably represents rare mRNAs expressed in only some brain cells. Therefore, most mRNA expressed in brain is either specific to brain or otherwise displays regulation. Rarer mRNA species tend to be larger than the more abundant species, and tend to be brain specific; the rarest, specific mRNAs average 5000 nucleotides in length. Ten percent of the clones hybridize to multiple mRNAs, some of which are expressed from small multigenic families. From these data we estimate that there are probably at most 30,000 distinct mRNA species expressed in the rat brain, the majority of which are uniquely expressed in the brain.

Differential gene expression in the gastrula of Xenopus laevis

Abstract: A modified cloning method designed to produce differential complementary DNA libraries permits the isolation of sequences that are present in the RNA population of any developmental stage or tissue, but are not present or are much less abundant in another stage or tissue. Selective complementary DNA cloning is especially useful when the differentially expressed RNA's are of low to moderate abundance in the cells in which they occur. A class of cytoplasmic polyadenylated RNA's differentially expressed in gastrula embryos of Xenopus laevis (DG RNA's) has been isolated. These DG RNA's occur very rarely or not at all in unfertilized eggs and blastulae, accumulate as the result of transcription before and during gastrulation, and, with some exceptions, decline in abundance as development proceeds. Many of these RNA molecules appear to be translated at the gastrula stage. Thus, DG RNA's may encode proteins that are important in the process of gastrulation.

Isolation of genomic clones containing the amdS gene of Aspergillus nidulans and their use in the analysis of structural and regulatory mutations.

Abstract: Previous analysis of the amdS gene of Aspergillus nidulans has identified multiple regulatory circuits mediated by trans-acting regulatory genes, cis-acting mutations have been identified and shown to specifically affect individual regulatory circuits. Fine-structure genetic mapping of the amdS regions showed that these cis-acting mutations occur in a complex controlling region adjacent to the amdS structural gene. The amdS gene was cloned by differential hybridization, using cDNA probes derived from a high-level-producing strain and from a strain with a large amdS deletion mutation. RNA blotting experiments showed that a single RNA species of 1,600 to 1,700 base pairs is transcribed from the amdS gene. DNA blotting experiments on a large number of amdS mutant strains, including deletions and translocations, allowed the genetic and physical maps of the gene to be correlated. The controlling region of the gene is situated at the 5' end of the gene and the direction of transcription is toward the centromere of chromosome III. The regulatory mutations in the controlling region were found to be due to small-scale alterations in the DNA rather than to large-scale rearrangements resulting in gene fusions.

Beta-globin gene inactivation by DNA translocation in gamma beta-thalassaemia.

Abstract: The beta-globin gene present on the deletion locus in a Dutch gamma beta-thalassaemic patient was found to be identical to the normal beta-globin gene with respect to DNA sequence and its transcription in HeLa cells. DNase I sensitivity and methylation experiments show that the affected beta-globin gene is present in an inactive configuration in vivo. This is the result of a translocation of a normally inactive locus next to the beta-globin gene on the affected chromosome, or the deletion of sequences which are normally required for the maintenance of the active state.

Signals regulating hepatitis B surface antigen transcription

Abstract: About 200 million people are chronic carriers of hepatitis B surface antigen (HBsAg), but since hepatitis B virus (HBV) cannot be propagated in vitro, HBsAg transcription has been studied only in cell lines containing HBV DNA integrated into chromosomes, and HBsAg-related mRNAs 2.0 to 2.5 kilobases (kb) long have been described1–4. We have analysed the transcripts produced in an infected chimpanzee liver and in a rat cell line containing HBV DNA5. In contrast to previous suppositions1,2 we report here that the major S gene transcript initiates close to the S gene, that is, within the ‘pre-S’ region6 and is processed/polyadenylated at a site situated within the core gene. The efficiency of processing/polyadenylation at this site varies between the chimpanzee liver and the rat cell line studied. The S gene promoter does not contain a TATA box but instead has a sequence homologous to that which positions the 5′ ends of the major simian virus 40 (SV40) late transcript7.

Regulation of yeast mating-type interconversion: feedback control of HO gene expression by the mating-type locus

Abstract: The ultimate product of yeast mating-type interconversion is a stable a/alpha diploid cell. A haploid cell carrying the HO gene gives rise to a diploid cell in a two-step process: first, the cell switches mating type as a result of genetic rearrangement (cassette substitution) catalyzed by HO; then, cells of opposite type mate to form a/alpha diploids. Mating-type interconversion does not occur in a/alpha diploids despite the presence of the HO gene. We have identified a plasmid carrying the HO gene by screening a yeast clone bank (constructed in vector YEp13) for plasmids that allow mating-type switching by ho cells. The yeast segment responsible for mating-type interconversion integrates by homology at the ho locus, thus confirming that it carries HO. Using the HO gene as a probe, we find that strains with an active mating-type interconversion system produce HO RNA, whereas a/alpha HO/HO cells do not and that this inhibition requires products of both the MATa1 and MATa2 genes. Thus, mating-type interconversion does not occur in a/alpha HO/HO cells because the HO gene product is not synthesized. These results demonstrate the following: (i) The mating-type locus, proposed on genetic grounds to be a regulatory locus, controls expression of an unlinked gene (HO) at the level of RNA production. (ii) The HO gene is under negative feedback control: its expression is inhibited after successful completion of diploidization (formation of a/alpha diploids).

alpha-skeletal and alpha-cardiac actin genes are coexpressed in adult human skeletal muscle and heart.

Abstract: We determined the actin isotypes encoded by 30 actin cDNA clones previously isolated from an adult human muscle cDNA library. Using 3' untranslated region probes derived from alpha-skeletal, beta- and gamma-actin cDNAs and from an alpha-cardiac actin genomic clone, we showed that 28 of the cDNAs correspond to alpha-skeletal actin transcripts. Unexpectedly, however, the remaining two cDNA clones proved to derive from alpha-cardiac actin mRNA. Sequence analysis confirmed that the two skeletal muscle alpha-cardiac actin cDNAs are derived from transcripts of the cloned alpha-cardiac actin gene. Direct measurements of actin isotype mRNA expression in human skeletal muscle showed that alpha-cardiac actin mRNA is expressed at 5% the level of alpha-skeletal actin. Furthermore, the alpha-cardiac actin gene expressed in skeletal muscle is the same gene which produces alpha-cardiac actin mRNA in the human heart. Of equal surprise, we found that alpha-skeletal actin mRNA accounts for about half of the total actin mRNA in adult heart. Comparison of total actin mRNA levels in adult skeletal muscle and adult heart revealed that the steady-state levels in skeletal muscle are about twofold greater, per microgram of total cellular RNA, than those in heart. Thus, in skeletal muscle and in heart, both of the sarcomeric actin mRNA isotypes are quite abundant transcripts. We conclude that alpha-skeletal and alpha-cardiac actin genes are coexpressed as an actin pair in human adult striated muscles. Since the smooth-muscle actins (aortic and stomach) and the cytoplasmic actins (beta and gamma) are known to be coexpressed in smooth muscle and nonmuscle cells, respectively, we postulate that coexpression of actin pairs may be a common feature of mammalian actin gene expression in all tissues.

Coordinate regulation of multiple histone mRNAs during the cell cycle in HeLa cells

Abstract: Core histone gene expression in HeLa S3 cells has been examined as a function of the cell cycle using cloned human histone gene probes. Total cellular histone mRNAs were analyzed by Northern blot analysis, and their relative abundance shown to be temporally coupled to DNA synthesis rates in S phase. The in vivo incorporation of 3H-uridine into at least fifteen heterologous histone mRNAs (in one hour pulse intervals at various times in the cell cycle), was monitored by hybrid selection. Hybridized RNAs were eluted and resolved electrophoretically to give both a quantitative and qualitative assay for multiple mRNA species. Maximal incorporation of 3H-uridine into histone mRNAs precedes their maximal accumulation, indicating that transcriptional regulation is predominant in early S phase. The turnover of histone mRNAs in late S occurs in the presence of a reduced apparent transcription rate, indicating that post-transcriptional regulation is predominant in late S. All the detected multiple histone mRNAs are coordinately regulated during the HeLa cell cycle.

Isolation and characterization of the human apolipoprotein A-I gene.

Abstract: We have recently shown that an inherited polymorphism occurring in the human apolipoprotein A-I (apo A-I) gene is related to decreased high density lipoprotein and apo A-I levels in the plasma of two patients with severe premature atherosclerosis. Analysis of the molecular basis of this polymorphism and its possible effects on apo A-I gene expression requires direct comparison of both normal and polymorphic apo A-I alleles. Here we report the isolation and characterization of the normal human apo A-I gene and we show that the gene is interrupted by three intervening sequences, IVS-1, IVS-2, and IVS-3, occurring in the 5' noncoding region of apo A-I mRNA, the mRNA sequence coding for the signal peptide of apo A-I, and the sequence coding for the mature protein, respectively. In addition, the nucleotide sequence analysis of the apo A-I gene allowed determination of the complete amino acid sequence of the primary translation product of apo A-I mRNA. This amino acid sequence consists of 267 residues including a 24-residue-long amino-terminal extension (preprosegment). Finally, we show that the apo A-I gene contains six 66-base-pair-long tandemly repeated DNA segments, which suggests that the gene may have evolved by intragenic duplication events.

Regulation of tubulin and actin mRNA production in rat brain: expression of a new beta-tubulin mRNA with development.

Abstract: The expression of alpha-tubulin, beta-tubulin, and actin mRNA during rat brain development has been examined by using specific cDNA clones and in vitro translation techniques. During brain maturation (0 to 80 days postnatal), these mRNA species undergo a significant decrease in abundance. The kinetics of this decrease varies between the cerebrum and the cerebellum. These mRNAs are most abundant in both tissues during week 1 postnatal, each representing 10 to 15% of total mRNA activity. Both alpha- and beta-tubulin mRNA content decreases by 90 to 95% in the cerebrum after day 11 postnatal, and 70 to 80% decreases in the cerebellum after day 16. Actin sequences also decrease but to a lesser extent in both tissues (i.e., 50%). These decreases coincide with the major developmental morphological changes (i.e., neurite extension) occurring during this postnatal period. These studies have also identified the appearance of a new 2.5-kilobase beta-tubulin mRNA species, which is more predominant in the cerebellar cytoplasm. The appearance of this form occurs at a time when the major 1.8-kilobase beta-tubulin mRNA levels are declining. The possibility that the tubulin multigene family is phenotypically expressed and then this expression responds to the morphological state of the nerve cells is discussed.

An MF alpha 1-SUC2 (alpha-factor-invertase) gene fusion for study of protein localization and gene expression in yeast

Abstract: The peptide mating pheromone alpha-factor and the hydrolytic enzyme invertase (beta-D-fructofuranoside fructohydrolase, EC 3.2.1.26) are processed from larger precursor proteins during their secretion from yeast cells (Saccharomyces cerevisiae). An in-frame fusion of the structural genes for these two proteins was constructed by connecting the 5'-flanking region and prepro-leader portion of the coding sequence of the alpha-factor gene (MF alpha 1) to a large fragment of the invertase gene (SUC2) lacking its 5'-flanking region and the coding information for the first four amino acids of its signal sequence. Sites that have been implicated in normal proteolytic processing of the alpha-factor precursor have been retained in this construction. The chimeric gene directs synthesis of a high level of active invertase that is secreted efficiently into the periplasmic space, permitting cell growth on sucrose-containing media. This extracellular invertase appears to contain no prepro-alpha-factor sequences. The initial intracellular product is, however, a hybrid protein that can be detected either by treatment of the cells with the drug tunicamycin or by blockage of secretion in a temperature-conditional secretion-defective mutant (sec18). Therefore, prior to its efficient proteolytic removal, the alpha-factor portion of the hybrid protein apparently provides the necessary information for efficient export of the substantially larger protein invertase. Similar to MF alpha 1, the MF alpha 1-SUC2 fusion is expressed in alpha haploids at levels 65-75 times higher than in a haploids or in a/alpha diploids; also, high-level expression is eliminated in mat alpha 1 mutants but not in mat alpha 2 mutants. Unlike expression of SUC2, expression of the fusion is not affected by glucose concentration. Hence, the 5'-flanking region present in the fusion (about 950 base pairs) is sufficient to confer alpha cell-specific expression to the hybrid gene.

Expression of a microinjected immunoglobulin gene in the spleen of transgenic mice

Abstract: Transgenic mice were produced by microinjection of a rearranged, functional immunoglobulin κ gene into fertilized mouse eggs and implantation of the microinjected embryos into foster mothers. Mice that integrated the injected gene were mated and the DNA, RNA and serum κ chains of their offspring were analysed. The data from offspring of three different transgenic mice indicate that the microinjected gene is expressed in the spleen, but not the liver of mice which inherited the injected gene.