Transcription Unit Of Gene Research Articles

A method for analyzing transcription using permeabilized cells

Brief exposure of Friend cells to a buffered hypotonic solution containing 1% Tween 80 caused permeabilization and allowed incorporation of [ 3H]UTP into RNA. The incorporation was inhibited 85–97% by 20 μg/ml actinomycin D and the reaction product was completely hydrolyzed by 0.1 m KOH. UMP incorporation was nearly linear for 60 min at 23°C; however, at 37°C it ceased after 15–20 min of rapid incorporation. The inhibition of UMP incorporation by 2 μg/ml α-amanitin was much greater at 23°C than at 37°C. The molecular weight of the RNA synthesized in permeabilized cells is broadly distributed with about 83% larger than 18 S. In vitro transcription of the mouse β-major globin gene was studied by hybridizing 32P-labeled nascent RNA to filter-bound DNA sequences representing this gene and its flanking regions. After induction by hexamethylene-bisacetamide, Friend cells exhibited more than fivefold increases in the rate of transcription for the β-major globin gene as compared to the uninduced control cells. Induction also caused an increase in the transcription rate of the 3′-flanking region located downstream from the poly(A) addition site. Thus, the primary transcription unit of β-major globin gene is essentially the same in permeabilized cells as that previously reported for nuclei isolated from the same cell line. In addition, permeabilized cells actively initiate RNA synthesis as determined by the incorporation of a thiol group at the 5′ initiating nucleotide, when synthesis was in the presence of [γ- S]-labeled nucleoside triphosphates. Permeabilized cells are about 7–11 times more active than isolated nuclei in the synthesis of both in vitro-initiated and total RNA.

The Yc and Ya subunits of rat liver glutathione S-transferases are the products of separate genes.

Rat liver glutathione S-transferases consist of binary combinations of three major classes of subunits designated as Ya (Mr = 25,600), Yb (Mr = 27,000) and Yc (Mr = 28,000). We have determined the nucleotide sequences of a cDNA insert in pGTR262 containing partial sequence of a Yc subunit. Sequence comparison with a Ya subunit cDNA clone pGTR261 revealed 70% nucleotide sequence homology and 65% amino acid sequence homology in the overlapping coding regions. Approximately 65% of the amino acid substitutions between these two subunits occur in clusters of two to eight residues. The 3' noncoding sequences of these two subunit cDNA clones are highly divergent in length and in sequences. The 3' noncoding region of pGTR262 cDNA contains open reading frames of 23, 39, and 13 amino acids which are in phase with the deduced Yc subunit sequences but interrupted by termination codons. By RNA blot hybridization analysis, we found that this Yc subunit sequence expression is tissue specific. It is expressed at a reduced level in kidney and testis as compared with liver and hardly at all in heart, lung, seminal vesicles, and spleen. We suggest that the mechanism for the tissue-specific expression of the Yc subunit of rat liver glutathione S-transferases may occur at or prior to the level of RNA processing. From the divergent DNA sequences between the Ya and Yc subunits reported in this communication and their differential induction upon acute phenobarbital administration reported by Pickett et al. (Pickett, C.B., Donohue, A. M., Lu, A. Y. H., and Hales, B. F. (1982) Arch. Biochem. Biophys. 215, 539-543) we propose that the Ya and Yc subunits of rat liver glutathione S-transferases are encoded by separate gene families or transcriptional units.

The absence of a human-specific ribosomal DNA transcription factor leads to nucleolar dominance in mouse greater than human hybrid cells.

The basis for nucleolar dominance in mouse-human cell hybrids which contained all of the mouse chromosomes but an incomplete set of human chromosomes (M greater than H) was examined at the molecular level. S1 mapping data showed that these cells had the expected levels of steady-state rRNA transcribed from mouse ribosomal gene (rDNA) transcription units but undetectable levels of rRNA derived from the human rDNA transcription templates that are also present. RNA polymerase I-dependent, cell-free transcription extracts were made from three hybrid lines and were found to be capable of transcribing cloned rDNA templates of mouse but not human origin. Partially purified human factors required for rDNA transcription in vitro were added to the M greater than H extracts. One fraction with almost no RNA polymerase I activity conferred on these hybrid cell extracts the ability to transcribe a human rDNA template. These rescue experiments suggested that this required human-specific rDNA transcription factor(s) was effectively absent from the lines we examined and could account for nucleolar dominance in M greater than H hybrid cells.

The absence of a human-specific ribosomal DNA transcription factor leads to nucleolar dominance in mouse greater than human hybrid cells.

The basis for nucleolar dominance in mouse-human cell hybrids which contained all of the mouse chromosomes but an incomplete set of human chromosomes (M greater than H) was examined at the molecular level. S1 mapping data showed that these cells had the expected levels of steady-state rRNA transcribed from mouse ribosomal gene (rDNA) transcription units but undetectable levels of rRNA derived from the human rDNA transcription templates that are also present. RNA polymerase I-dependent, cell-free transcription extracts were made from three hybrid lines and were found to be capable of transcribing cloned rDNA templates of mouse but not human origin. Partially purified human factors required for rDNA transcription in vitro were added to the M greater than H extracts. One fraction with almost no RNA polymerase I activity conferred on these hybrid cell extracts the ability to transcribe a human rDNA template. These rescue experiments suggested that this required human-specific rDNA transcription factor(s) was effectively absent from the lines we examined and could account for nucleolar dominance in M greater than H hybrid cells.

Chromatin organization of the 87A7 heat shock locus of Drosophila melanogaster

We have examined the chromatin structure of the hsp 70 gene complex at the 87A7 heat shock locus of Drosophila melanogaster. Our results indicate that this locus has a complex chromatin organization. Heat induction causes highly specific alterations in the chromatin throughout the locus. There are major changes within the heat shock gene transcription units, and in both the upstream and downstream flanking spacers.

Unusual heterogeneity of the 5'-termini of human adenovirus type 2 early region E2 mRNA.

The 5'-terminal structures of human adenovirus type 2 (Ad2) early region 2 (E2) mRNA were investigated. The E2 transcription unit has several interesting properties, including the presence of a TATA-like box that matches the consensus sequence poorly, delayed transcription during early stages of infection, and a switch in promoter recognition late after infection. E2-specific RNA, 5'-labeled in vitro to high specific activity was analyzed. Purified E2 mRNA was digested with RNase A or RNase T1 and the resulting oligonucleotides were resolved by two dimensional paper electrophoresis-homochromatography. Remarkably, as many as sixteen 5'-terminal RNase A oligonucleotides were identified and their sequences were deduced. The most common 5'-termini in the RNase A digest were p(m6)AmCp, p(m6)AmA(m)Cp, pGmA(m)Cp, and p(m6)AmG(m)Cp. Two RNase A oligonucleotides originated from the E4 promoter region, consistent with electron microscopic observations. The sequence encoding these potential initiation sites covered about 90 nucleotides. Eleven of the sequences of the 5'-terminal RNase A oligonucleotides were aligned with the Ad2 DNA sequence in the Ad2 E2 promoter region. If the heterogeneous termini in the E2 promoter region were generated by a process of transcription initiation, their existence cannot be explained by stuttering of RNA polymerase II. This suggests that the transcription of Ad early region 2 has features which differ from those of other Ad2 early gene transcription units. Perhaps this is due to the absence of a conventional TATA box which is believed to position the initiation site. Alternatively, it is conceivable that the E2 promoter represents an alternate class of RNA polymerase II promoters containing different signals with different requirements for activation and/or that an E1A gene product modifies transcription initiation.

Seminal vesicle secretion IV gene: allelic difference due to a series of 20-base-pair direct tandem repeats within an intron.

The rat seminal vesicle secretion IV (SVS IV) gene was isolated from a lambda Charon 4A library. The SVS IV gene transcription unit was found to be on one 3.3-kilobase (kb) EcoRI fragment. Restriction mapping and DNA sequence analysis demonstrated that the entire length of the SVS IV transcription unit is 1,930 base pairs (bp) and contains two introns. The 3.3-kb EcoRI fragment contains 144 bp of 5'-flanking region. At -113 bp from the presumed transcription initiation site an interesting structure with perfect dyad symmetry is noted. In another lambda clone, a 3.5-kb EcoRI fragment was isolated that contains the SVS IV gene and was shown to be identical to the 3.3-kb EcoRI fragment except for 180 bp of DNA in the second intron. The extra DNA consists of several (8-10) 20-bp tandem repeats flanked on each side by seven or eight copies of this same 20-bp repeat. Fisher X Sprague-Dawley hybrid rats, which contain both the EcoRI 3.5-kb form and the 3.3-kb form of the SVS IV gene, were crossed with each other. Analysis of the F1 generation demonstrated that the presence or absence of the 180-bp intronic insertion in the SVS IV gene defines an allelic difference. This report also presents the DNA sequence of the transcription unit and flanking regions of the SVS IV gene.

The association of transcriptionally active genes with the nuclear matrix of the chicken oviduct.

Eucaryotic DNA is organized into a series of supercoiled loops that are anchored to the nuclear matrix. When these DNA loops are cleaved by endonucleases, the DNA sequences which remain associated with the nuclear matrix can be recovered and analyzed for their content of specific genes. Using restriction endonucleases to cleave the loops, we demonstrate that ovalbumin and conalbumin gene sequences are preferentially associated with the nuclear matrix of hen oviduct cells but not with the nuclear matrix of hen brain cells. Furthermore, we determined that several regions of the ovalbumin gene were independently attached to the nuclear matrix of hen oviduct cells. This included sequences located 3.8 kb downstream from the 3' end of the ovalbumin gene transcription unit. To determine whether the nuclear matrix association of the ovalbumin gene was regulated by hormones, we examined the oviduct cells of chicks that underwent primary estrogen stimulation, estrogen withdrawal and secondary estrogen stimulation. Ovalbumin gene sequences selectively dissociated from the chick oviduct nuclear matrix during estrogen withdrawal and reassociated with the nuclear matrix following restimulation.

Plasminogen Activator: Analysis of Enzyme Induction by Ultraviolet Irradiation Mapping

Ultraviolet irradiation mapping techniques have previously been used to study the organization of eucaryotic gene classes and transcription units. We used the same method to probe some regulatory phenomena observed in the induction of plasminogen activator (PA) biosynthesis: PA synthesis in chicken embryo fibroblasts is induced by tumor-promoting phorbol esters and by retinoic acid; furthermore, PA induction by phorbol esters is synergistic with transformation, being 10- to 20-fold greater in virus-transformed cells than in normal cells. We found that the ultraviolet irradiation inactivation cross sections for PA induction by phorbol esters and by retinoate differed significantly, suggesting that these agents induce PA biosynthesis by different mechanisms. On the other hand, the ultraviolet irradiation sensitivity of phorbol ester induction in normal chicken embryo fibroblasts was the same as in transformed cells, indicating that the synergism of transformation and phorbol esters is probably not due to different pathways of PA induction.

Plasminogen activator: analysis of enzyme induction by ultraviolet irradiation mapping.

Ultraviolet irradiation mapping techniques have previously been used to study the organization of eucaryotic gene classes and transcription units. We used the same method to probe some regulatory phenomena observed in the induction of plasminogen activator (PA) biosynthesis: PA synthesis in chicken embryo fibroblasts is induced by tumor-promoting phorbol esters and by retinoic acid; furthermore, PA induction by phorbol esters is synergistic with transformation, being 10- to 20-fold greater in virus-transformed cells than in normal cells. We found that the ultraviolet irradiation inactivation cross sections for PA induction by phorbol esters and by retinoate differed significantly, suggesting that these agents induce PA biosynthesis by different mechanisms. On the other hand, the ultraviolet irradiation sensitivity of phorbol ester induction in normal chicken embryo fibroblasts was the same as in transformed cells, indicating that the synergism of transformation and phorbol esters is probably not due to different pathways of PA induction.

The primary transcription unit of the mouse β-Major globin gene

The transcriptional unit boundaries of the mouse β-major globin gene have been investigated in DMSO-induced murine erythroleukemia cells using mainly labeled RNA produced by in vitro transcription in isolated nuclei. Restriction fragments of a genomic β-major globin clone (Tilghman et al. 1977) representing regions of the 5′ and 3′ portions of the mRNA sequence, the large intervening sequence, as well as regions before the cap site and beyond the poly(A) site of the mRNA were recloned for use in DNA excess hybridization experiments. Equimolar amounts of labeled nascent RNA hybridized to the fragment encoding the capped 5′ end of the mRNA and to fragments extending at least 1000 nucleotides beyond the poly(A) addition site. The existence of continuous transcription throughout this region was shown by: samples of labeled RNA each specifically complementary to one of the globin region fragments all bound to a single strand, presumably the coding strand of the separated strands of the β-globin genomic clone; specific T1 oligonucleotides diagnostic of the region immediately following the poly(A) site of the nuclear RNA were identified in hnRNA labeled in isolated nuclei; the shortest labeled nascent RNA chains bound to the fragment containing the cap site and increasingly longer chains bound in the ordered set of fragments as a function of distance from the cap site. These results suggest the necessity for an endonucleolytic cleavage of a primary transcript followed by polyadenylation in the process of creating the 3′ end of this cellular mRNA, as has previously been demonstrated for every virus mRNA so far studied (Nevins and Darnell 1978; Ford and Hsu 1978; Fraser et al. 1979; Nevins et al. 1980). An analysis of the accumulation of the different RNA sequences labeled in vivo demonstrated a confinement of the sequences transcribed past the poly(A) site exclusively to the nucleus and indicated an even higher turnover rate for these than for the intervening sequences. In addition to the equimolar amounts of labeled nascent RNA complementary to DNA fragments from the cap site through and past the poly(A) site, a smaller amount of labeled RNA hybridized reproducibly to DNA fragments upstream from the cap site. Although this RNA was complementary to the coding strand, it did not appear to be part of the majority of the globin primary transcripts based on size analysis and it was present at only 20% or less of the molarity of the RNA from the globin coding regions.