![Autoradiograph of the RNA-protected 3' end 32P-labeled DNA fragments after digestion with Si nuclease. The DNA was linearized as described for Fig. 8 and labeled at the 3' termini with [a-32P]dGTP as described in Materials and Methods. S1 nuclease digestion and electrophoresis were similar to the description for Fig. 8. The open arrowhead indicates the main DNA fragment protected from S1 nuclease digestion by the RNAs.](https://www.researchgate.net/profile/Guang-Jer-Wu-2/publication/19788224/figure/fig2/AS:667801782063113@1536227760386/Autoradiograph-of-the-RNA-protected-3-end-32P-labeled-DNA-fragments-after-digestion-with_Q320.jpg)
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Organization of multiple regulatory elements in the control region of the adenovirus type 2-specific VARNA1 gene: fine mapping with linker-scanning mutants
Abstract and Figures
The adenovirus type 2-specific virus-associated RNA 1 (VARNA1) gene is transcribed by eucaryotic RNA polymerase III. Previous studies using deletion mutants for transcription have shown that the VARNA1 gene has a large control region which is composed of several regulatory elements. Twenty-five exact linker-scanning mutations in the control region, from -33 to +77, of this gene were used for definition of the number and boundaries of these elements. The effects of these mutations on transcription and competition for transcription factors in human KB cell extracts revealed five positive regulatory elements. The essential element, which coincided with the B block, was absolutely required for both transcription and formation of stable complexes. A second element, which included the A block, was also required for both transcription and formation of stable complexes. Although this element is not as essential as the B-block element, together with the B-block element it may be necessary for formation of the most basal form of transcription machinery. Therefore, these two elements are the promoter elements in this gene. In addition, one possible element in the interblock region and two elements in the 5' flanking region were also required for efficient transcription, but they were moderately required for formation of stable complexes. Transcription of these mutants and the wild-type gene using an extract of 293 cells was stimulated at least threefold over that with the KB cell extract, as expected. Similar regulatory elements of this gene were revealed, however, when the 293 cell extract was used for transcription of these mutants, suggesting that the E1A-mediated specific transcription factors act on the transcription machinery in a sequence-nonspecific manner.
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... First, we should test the effect of mutations in the six conserved sites of the huMETCAM/MUC18 on in vitro cell-cell aggregation and cell-extracellular matrix adhesion and on in vivo tumorigenesis and metastasis of human cancer cells. The N-glycosylation site can be point mutated from Asn to Ala or Gln [122,123] or may be linker-scanning mutated by replacing the three codons (the nine nucleotide sequence) with a nine bp oligonucleotide containing a unique restriction site sequence [124]. Both kinds of mutation are better than deletion mutations, since they do not change the relative physical location of the mutated sequences and thus the phenotype of the mutant is directly related to the substituted sequence without the complicated influence of the added sequences from the surrounding region of interest. ...
... Both kinds of mutation are better than deletion mutations, since they do not change the relative physical location of the mutated sequences and thus the phenotype of the mutant is directly related to the substituted sequence without the complicated influence of the added sequences from the surrounding region of interest. The linker-scanning mutations from our experience are superior to the point mutations because they usually manifest a more dramatic phenotype [124]. ...
... The VA RNA genes are regulated by intragenic control elements similar to tRNA genes [7,8] . The locations and functions of these elements , known as box A and box B, have been thoroughly mapped by mutational ana lysis91011. For example, in Ad2 VA RNAI, the box A (position +17 to +27) and box B (position +62 to +72) have been shown to be essential for VA RNA transcription (Figure 1) [10] . ...
Adenovirus type 5 encodes two short, highly structured noncoding RNAs, the virus-associated (VA) RNAI and VA RNAII. These RNAs are expressed in large amounts late during a lytic infection. Early studies established an important role for VA RNAI in maintaining efficient translation in late virus-infected cells by blocking activation of the key interferon-induced PKR protein kinase. More recent studies have demonstrated that the VA RNAs also target the RNAi/miRNA pathway. Collectively, available data suggest that the VA RNAs are multifunctional RNAs suppressing the activity of three dsRNA-sensing enzyme systems in human cells. Here, the known functions of the VA RNAs are summarized and the interplay between VA RNA expression and the activity of the interferon and RNAi pathways are discussed in more detail.
We have studied the mechanism by which 5'-flanking sequences modulate the in vitro transcription of eucaryotic tRNA genes. Using deletion and linker substitution mutagenesis, we have found that the 5'-flanking sequences responsible for the different in vitro transcription levels of three Drosophila tRNA5Asn genes are contained within a discrete region centered 22 nucleotides upstream from the transcription initiation site. In conjunction with the A-box intragenic control region, this upstream transcription-modulatory region functions in the selection mechanism for the site of transcription initiation. Since the transcription-modulatory region directs the position of the start site and the actual sequence of the transcription-modulatory region determines the level of tRNAAsn gene transcription, the possibility is raised that the transcription-modulatory region directs a transcription initiation event similar to open complex formation at procaryotic promoters.
Human transcription factor IIIC (TFIIIC) is an initiation factor required for the in vitro transcription of 5 S RNA, tRNA, and adenovirus viral-associated (VA) RNA genes by RNA polymerase III. A TFIIIC activity which complemented purified TFIIIB and RNA polymerase III fractions for VA transcription was highly purified from cultured HeLa cells. This activity copurified through all chromatographic procedures, including B-block oligodeoxynucleotide affinity chromatography, with the two forms of TFIIIC detected by gel mobility shift assays with the VA gene (Hoeffler, W.K., Kovelman, R., and Roeder, R.G. (1988) Cell 53, 907–920). Both specific binding activity to the VAI gene and TFIIIC transcription activity were inhibited by the alkylating agents diisopropyl fluorophosphate, N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), and N-ethylmaleimide, and to a lesser extent by N α-p-tosyl-L-lysine chloromethyl ketone, whereas neither activity was inhibited by phenylmethylsulfonyl fluoride. These data suggest further that the DNA binding and transcription assays scored the same protein(s). TPCK and N-ethylmaleimide inactivated TFIIIC solely through thiol group modification, since prior modification with the reversible thiol reagent 2,2′-dithiopyridine prevented permanent inactivation. The involvement of reduced thiol groups in the specific binding of TFIIIC to the VAI gene was further indicated by an increase in TFIIIC binding activity upon addition of dithiothreitol. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that a Mr = 126,000 polypeptide both eluted from a B-block oligodeoxynucleotide affinity column with the DNA binding and transcription activities of TFIIIC and was specifically cross-linked by UV to a 5-bromo-2-deoxynucleotide-substituted B-block oligodeoxynucleotide. The near identity of the TFIIIC molecular weight determined by gel filtration on SOTA Phase GF 200 (Mr = 140,000) suggests that TFIIIC in solution (in the presence of 0.3 M NaCl at pH 7.0) consists of a single polypeptide which is fairly globular in nature.
Once a preinitiation complex has formed on a yeast tRNA gene, RNA chain initiation requires a further 5 min at 22°C (half-life ~2 min).1 During this period, three successive steps occur: recruitment of pol III, melting of the DNA helix, and initiation of RNA synthesis. Recruitment and melting are the slow steps, whereas the subsequent synthesis of a seventeen base transcript is completed within 10 seconds.1
The promoters of most class III genes include discontinuous intragenic structures, termed internal control regions (ICRs), that are composed of essential sequence blocks separated by nonessential nucleotides. The ICRs of 5S rRNA genes are sometimes referred to as type I. These comprise two functional domains: an Ablock and a second domain consisting of an intermediate element and a C-block. Most class III genes, including tRNA, VA, Alu, EBER, 7SL, 4.5S, B1, and B2 genes, have type II ICRs: these again have two domains, an A-block and a B-block. The Ablocks of types I and II are homologous and can substitute for one another in Xenopus, 1 although not in Neurospora.2 The A-block is located much further from the start site in type I than it is in type II promoters. As well as the ICR, extragenic sequences can also affect the strength of type I and II promoters. However, substitutions in the extragenic regions are generally well tolerated, unlike mutations in the ICR. In contrast, with type III promoters, such as those of the vertebrate U6 and 7SK genes, transcription is independent of intragenic elements and is dictated solely by 5′ flanking regions.3–8 A schematic illustration of the three types of class III promoter is provided in Figure 2.1.
Adenoviruses were first isolated by Rowe and co-workers from human adenoid tissues—hence the name adenoviruses. These are nonenveloped DNA viruses, composed of only DNA and proteins, and are widespread in nature. Forty-one of the 93 strains identified so far are of human origin, while the rest have been isolated from monkeys, dogs, cattle, rodents, and birds.1–3 Clinical studies have shown that these viruses can cause a variety of diseases in humans, most of which involve the respiratory tract, the eye, and the gastrointestinal tract. For the most part, these infections lead to self-limited illnesses or latent persistent infections and are usually followed by complete recovery. Adenoviruses can maintain inapparent infections within host animals for months or even years. Lymphoid cells may serve as reservoirs for these persistent infections. Several members of this group can also induce malignant tumors in newborn rodents and therefore adenoviruses are classified as members of the DNA tumor virus group. All the members of this group tested so far have been shown to transform rodent cells in culture.4 Despite their tumorigenic potential in experimental models, these viruses have not been shown to be tumorigenic in humans. Adenoviruses can be grown to high titers in a variety of cultured mammalian cells. Human cells of epithelial origin are best suited for growing human adenoviruses. Because of the small size of their genome, the ease with which they can be grown in rapidly growing cultured cells, and their ability to transform rodent cells in vitro, adenoviruses have served as important model systems to study the regulation of eukaryotic gene expression and virus-host-cell interactions.
The aberrant expression of cell adhesion molecules (CAMs) is correlated with the malignant progression of many tumors. MUC18/CD146/A32/MelCAM/S-endo, an integral membrane glycoprotein, is a CAM in the immunoglobulin gene superfamily. MUC18 has often been mistaken as a mucin because of the misleading nomenclature. Based on its biological functions and other known biochemical properties, we propose to re-name it as METCAM (metastasis CAM). METCAM/MUC18 expression has a very interesting effect on tumor formation and metastasis. The overexpression of METCAM/MUC18 has been correlated with the malignant progression of human melanoma and human prostate cancer. In melanoma, the ectopic over-expression of METCAM/MUC18 has no effect on tumorigenesis, but augments the metastasis of cancer cells. In prostate cancer, the ectopic over-expression of human METCAM/MUC18 has an even greater effect, increasing tumorigenesis and initiating the metastasis of cancer cells. However, an opposite effect of METCAM/MUC18 on tumorigenesis and metastasis of breast cancer, some mouse melanoma cell lines, and perhaps haemangioma and nasopharygeal carcinoma has also been suggested. Taken together, we suggest that the different effect of METCAM/MUC18 on tumor formation and metastasis is dependent on the intrinsic properties of each tumor cell line. This paper will review previous experiments and results and present some possible mechanisms of METCAM/MUC18- mediated tumorigenesis and metastasis for future studies.
Two genes for virus-associated VA RNAs in the human adenovirus type 7 (Ad7) genome are compared to Ad2, Ad4, Ad5, simian VA RNAs, and pol III transcripts of Epstein-Barr virus. The newly identified VA RNA-encoding genes of Ad7 contain two relatively conserved intragenic promoter (elements A and B), the double-stranded RNA-dependent protein kinase (PK) active site-binding domain and transcriptional terminators. The conserved features extend to the VA RNA genes of simian and avian origin.
We constructed mutants with a deletion of either half of the 18 base-pair B block palindrome in the VARNA1 gene, mutants with different intra-palindromic spacings, a complete set of mutants with single base substitutions, and mutants with double and triple base substitutions in the palindrome. The transcription efficiencies of these mutants were determined in human KB cell-free cytoplasmic S100 extracts. The relative competing strength of each mutant, as determined by a sequential competition experiment, was used to assess each mutant's ability to sequester factors into formation of a stable preinitiation complex. The ability of each mutant to assemble transcriptionally active preinitiation complexes was also determined by direct transcription of the isolated complexes. Finally, the ability of each mutant to interact with the transcription factor(s) TFIIIC and form a distinct gel-resolved complex was also determined. From the results of the above assays, we concluded that the two seemingly identical halves of the palindrome did not contribute equally to transcription, or to assembly of the functional preinitiation complex, nor to interaction with TFIIIC. The anterior half (B1) of the B block palindrome, which is proximal to the A block promoter element, played a stronger role in transcription and in assembly of the functional preinitiation complex than the posterior half (B2) of the palindrome. Consistent with this observation, the point mutations in four base-pairs, GTTC, from +60 to +63 in the anterior half of the B block palindrome, has the most severe effect on transcription. In contrast, we showed that the central sequence and the posterior half (B2) played a stronger role than the anterior half (B1) of the B block palindrome in the interaction of the promoter with TFIIIC. This was corroborated by the observation that base substitutions in the central four base-pair sequence of the palindrome, TCGA, from +62 to +65, had the most severe effect on interaction with TFIIIC, and that mutations in most of the sequences in the posterior half of the B block palindrome had more drastic effects than mutations in the anterior half of the palindrome in this interaction. Furthermore, the spacing between the two halves of the B block palindrome had a drastic effect on the overall transcription efficiency and the interaction of the promoter with TFIIIC, suggesting that the interaction between the two halves of the B block palindrome is not only essential, but also synergistic for the interaction with TFIIIC as well as the assembly of a transcriptionally active preinitiation complex and efficient transcription.
A cell-free system developed from human KB cells was used to transcribe 5.5S RNA from deproteinized adenovirus DNA in vitro. The cell-free RNA synthesis is dependent upon exogenous templates, ribonucleoside triphosphates, and cell-free postmitochondrial supernatant of human KB cells. The synthesis of 5.5S RNA is inhibited only by high levels of alpha-amanitin; therefore it is carried out by RNA polymerase III. The rate of synthesis was linear for at least 2 hr, indicating reinitiation. The 5.5S RNA synthesized in vitro is similar to the corresponding in vivo RNA in size, sequence, and coding region on adenovirus type 2 DNA. In this report is demonstrated in vitro synthesis of a facsimile of an in vivo transcript directed by deproteinized DNA in a mammalian cell-free postmitochondrial supernatant system.
VA-RNA I is a low molecular weight RNA produced in large amounts in cells infected with adenoviruses. The 3' terminus of this RNA may represent a transcription termination site. We have demonstrated that this RNA occurs in infected cells in several forms which differ in the number of uridylic acid residues at the 3' ends. The nucleotide sequence of a DNA fragment overlapping the 3' end of VA-RNA I has been determined. The DNA could encode up to 4 uridylic acid residues at the 3' end of the RNA. The DNA sequence shows some similarity to known transcription termination sequences in prokaryotic systems.
DNA preparations from about hundred randomly selected clones containing, mouse DNA fragments were screened for the existence of sequences complementary to long double-stranded regions of pre-mRNA able to snap back after melting (dsRNA-B). Many clones containing such sequences were found. The cloned sequences can be subdivided im;o three groups: (1) those complementary to about a half (at least to 30–40%) of the total dsRNA, designated as sequences B1; (2) those complementary to a part of sequence B1; and (3) sequences complementary to about a quarter (at least to 15%) of the total dsRNA referred to as sequence B2. The size of DNA sequence complementary to dsRNA is about 400 base pairs.
Melting experiments with hybrids show that the members of B1 family are very similar if not identical, while the divergence among B2 sequences is higher, but still the number of substitutions does not exceed 9% of bases. Thus, the major part of dsRNA-B consists of a small number of highly abundant sequences as was suggested earlier on the basis of renaturation kinetics /1–3/. Sequences B1 and B2 are represented by many copies in the mouse genome and in pre-mRNA, and many of them probably do not form hairpin-like structures.
The nucleotide sequence of the region of the Epstein-Barr virus genome that specifies two small ribonucleic acids (RNAs),
EBER 1 and EBER 2, has been determined. Both of these RNAs are encoded by the right-hand 1,000 base pairs of the EcoRI J fragment
of EBV deoxyribonucleic acid. EBER 1 is 166 (167) nucleotides long and EBER 2 is 172 +/- 1 nucleotides long; the heterogeneity
resides at the 3' termini. The EBER genes are separated by 161 base pairs and are transcribed from the same deoxyribonucleic
acid strand. In vitro, both EBER genes can be transcribed by RNA polymerase III; sequences homologous to previously identified
RNA polymerase III intragenic transcription control regions are present. Striking similarities are therefore apparent both
between the EBERs and the two adenovirus-associated RNAs, VAI and VAII, and between the regions of the two viral genomes that
specify these small RNAs. We have shown that VAII RNA as well as VAI RNA and the EBERs exist in ribonucleoprotein complexes
which are precipitable by anti-La antibodies associated with systemic lupus erythematosus. Finally, we have demonstrated that
the binding of protein(s) from uninfected cells confers antigenicity on each of the four virus-encoded small RNAs.
A new method for determining nucleotide sequences in DNA is described. It is similar to the "plus and minus" method [Sanger, F. & Coulson, A. R. (1975) J. Mol. Biol. 94, 441-448] but makes use of the 2',3'-dideoxy and arabinonucleoside analogues of the normal deoxynucleoside triphosphates, which act as specific chain-terminating inhibitors of DNA polymerase. The technique has been applied to the DNA of bacteriophage varphiX174 and is more rapid and more accurate than either the plus or the minus method.