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PubMed1994

Autoantigen Ku protein is involved in DNA binding proteins which recognize the U5 repressive element of human T-cell leukemia virus type I long terminal repeat.

We have identified and analyzed a 27-nucleotide sequence (U5 repressive element, designated as U5RE) at the U5 region of the human T-cell leukemia virus type I (HTLV-I) long terminal repeat (LTR) which is required for HTLV-I basal transcriptional repression. The basal promoter strength of constructs that contained deletions in the U5 region of the LTR was analyzed by chloramphenicol acetyltransferase (CAT) assays following transfection of HeLa cells or Jurkat T-cells in the presence or absence of viral transactivator tax protein. We consistently observed a 2- to 5-fold increase in basal promoter activity when sequences between +277 to +306 were deleted. In vivo competition experiments suggested that the U5 DNA fragment from +269 to +295 contains a functional repressive element (U5RE). Using gel mobility shift assays, we have purified a highly enriched fraction that could specifically bind U5RE. This DNA affinity column fraction contained three major detectable proteins on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with silver staining: 110-, 80- and 70-kDa proteins. The 110-kDa protein appeared to be a novel DNA-binding protein whose characteristics are still obscure, while the 70- and 80-kDa proteins were shown to be related to the human autoantigen Ku, the Ku (p70/p80) complex, as demonstrated by amino acid sequencing and immunological analyses. As Ku is known to be involved in transcriptional regulation, the specific interaction of Ku with U5RE raises intriguing possibilities for its function in HTLV-I basal transcriptional repression.

Amino Acid Sequence↗

PubMed1995

Transcriptional silencing of human Alu sequences and inhibition of protein binding in the box B regulatory elements by 5'-CG-3' methylation.

In earlier work, we demonstrated that 5'-CG-3' methylation inhibits the transcriptional activity of human Alu elements associated with the alpha 1-globin and the angiogenin genes in a cell-free transcription system from HeLa nuclear extracts. These studies have been extended to different Alu sequences and to investigations on the mechanism involved in transcriptional silencing by methylation. By comparing the results of DNase I and dimethyl sulfate (DMS) in vitro footprinting on a consensus sequence in the RNA polymerase III promoter control B region between the unmethylated and the 5'-CG-3' methylated B box, evidence has been adduced for effects of 5'-CG-3' methylation on the interaction of specific nuclear proteins with DNA sequences in the B control region of the Alu elements. These results are consistent with the interpretation that the 5'-CG-3' methylation interferes with the binding of proteins that are essential for the function of the B control region in these RNA polymerase III-transcribed elements, and that promoter methylation thus inhibits transcription.

Base Sequence↗

PubMed1995

The trans-inhibitory Rep78 protein of adeno-associated virus binds to TAR region DNA of the human immunodeficiency virus type 1 long terminal repeat.

The large rep gene products, Rep78 and Rep68, of adeno-associated virus (AAV) are pleiotropic effector proteins which are required for AAV DNA replication and the trans-regulation of AAV gene expression. Apart from these essential functions prerequisite for the life cycle of AAV, these rep products are able to inhibit the replication and gene expression of human immunodeficiency virus type 1 (HIV-1) and a number of DNA viruses. Here, it is demonstrated that Rep78, as a chimeric with the maltose binding protein, directly binds the full-length HIV-1 long terminal repeat (LTR), and to a subset of these sequences containing the trans-activation response (TAR) sequence as DNA. These interactions, an effector protein physically binding a target promoter, suggest a direct mechanism of action for Rep78 inhibition. Furthermore, competitive binding studies between the TAR region and the full-length HIV-LTR, strongly suggested that another site(s) within the LTR was also bound by Rep78. Finally, as Rep78 binding is also believed to be affected by secondary structure within the DNA, it was found that Rep78 preferentially binds with HIV-LTR sequences with promoted secondary structure generated by heat denaturation and rapid cooling.

Base Sequence↗

PubMed1989

Expression of synthetic calcitonin genes in plasmid vectors containing tandemly repeated non-overlapping ribosome binding sites.

1. Expression plasmids containing non-overlapping tandemly repeated ribosome binding sites (RBS) were constructed in order to stabilize mRNA and enhance translation. 2. Two synthetic genes (human calcitonin tetramer gene and a fusion gene human gamma-interferon-human calcitonin) were cloned in these vectors and the effect of multiplicity of Shine-Dalgarno (S/D) sequence on heterologous gene expression was studied. 3. It was found that duplication and triplication of RBS had no effect on the stability of mRNA but led to a strong decrease in the level of recombinant protein and mRNA in the cell. 4. Plasmids bearing four times repeated S/D sequences gave longer-lived mRNAs and maintained a level of protein and mRNA very close to the values obtained with a single S/D containing plasmids.

Base Sequence↗

PubMed1989

Structure of vaccinia virus early promoters.

Functional elements of a vaccinia virus early promoter were characterized by making a complete set of single nucleotide substitutions, as well as more complex mutations, and assaying their effects on gene expression. Synthetic oligonucleotides, based primarily on the sequence of the 7.5-kD early promoter, were inserted into a plasmid vector containing the lacZ gene of Escherichia coli flanked by sequences from the thymidine kinase (TK) gene of vaccinia virus. The lacZ gene, under control of the synthetic promoter, was introduced into the vaccinia virus genome at the TK locus by homologous recombination, and each of the 331 different recombinant viruses thus obtained was assayed for beta-galactosidase expression. The relative amounts and precise 5' ends of lacZ mRNAs specified by a subset of the recombinants were determined by primer extension. Many promoters were tested for their ability to direct specific transcription in vitro. A generally good correlation was noted between measurements of promoter strength estimated by beta-galactosidase expression, primer extension of in vivo mRNA and transcription in vitro. A relatively simple picture emerged from the analysis. The early promoter consists of a 16 base-pair critical region, in which most single nucleotide substitutions have a major effect on expression, separated by 11 base-pairs of a less critical T-rich sequence from a seven base-pair region within which initiation with a purine usually occurs. For the critical region of the 7.5-kD promoter, AAAAgTaGAAAataTA, any substitution of an upper-case nucleotide reduced expression, usually drastically, whereas certain substitutions of lower-case nucleotides maintained or significantly enhanced expression. On the basis of this analysis, the wide range of activities of natural promoters could be attributed to the presence of one or more non-optimal nucleotides in the critical region. Moreover, single nucleotide substitutions in such promoters had the predicted enhancing effects. Most mutations in the critical region of the 7.5-kD promoter behaved independently, but some nucleotide substitutions compensated for potentially detrimental nucleotides at other positions. Promoters substantially stronger than any natural ones examined were constructed by combining several up-mutations within the critical region of the 7.5-kD promoter and by repeating the critical region sequence. Like the TATA box of eukaryotic RNA polymerase II promoters, the critical region specifies the site of transcriptional initiation.

Base Sequence↗

PubMed1992

Promoter sequence containing (CT)n.(GA)n repeats is critical for the formation of the DNase I hypersensitive sites in the Drosophila hsp26 gene.

We have analyzed P-element-transformed lines carrying hsp26/lacZ transgenes with various deletions and substitutions within the Drosophila melanogaster hsp26 promoter region in order to identify the sequences required for the formation of the DNase I hypersensitive sites (DH sites). DH sites are generally found associated with promoters and enhancer elements of active and inducible eukaryotic genes, and are thought to be nucleosome-free regions of DNA that interact with regulatory proteins and the transcriptional machinery. There are two major DH sites located within the promoter region of the hsp26 gene, centered at -50 and at -350 (relative to the hsp26 transcription start site). The sequences from -135 to -85, which contain (CT)n.(GA)n repeats, contribute significantly to the formation of the DH sites in the hsp26 promoter region. Deletion or substitution of this (CT)n region drastically reduces the accessibility of the DNA at these sites to DNase I. This reduction in accessibility was quantified by measuring the susceptibility of the DNA within nuclei to cleavage at a restriction site within the DH site. In addition to the (CT)n region and the promoter at -85 to +11 (region P), one of two other regions must be present for effective creation of the DH sites: sequences between -351 and -135 (region A), or sequences between +11 and +632 (region D). Disruption of the wild-type chromatin structure, as assayed by the loss of accessibility to the DH sites, is correlated with a decrease in inducible transcriptional activity, even when the TATA box and heat shock regulatory elements are present in their normal positions.

Animals↗

PubMed1992

DNA sequence analysis of 66 kb of the human MHC class II region encoding a cluster of genes for antigen processing.

The genomic sequence of a 66,109 bp long region within the human MHC has been determined by manual and automated DNA sequencing. From cDNA mapping and sequencing data it is known that this region contains a cluster of at least four genes that are believed to be involved in antigen processing. Here, we describe the genomic organization of these genes, which comprise two proteasome-related genes (LMP2 and LMP7), thought to be involved in the proteolytic degradation of cytoplasmic antigens and two ABC transporter genes (TAP1 and TAP2), thought to be involved in pumping of the degraded peptides across the endoplasmic reticulum membrane. Analysis of the sequence homology and the intron/exon structures of the corresponding genes suggests that one gene pair arose by duplication from the other. Comparison of the available sequence data from other organisms shows striking conservation (70 to 84%) of this gene cluster in human, mouse and rat. The presence of several potential interferon stimulated response elements (ISREs) is in agreement with the experimentally observed up-regulation of these genes with gamma-interferon.

Amino Acid Sequence↗

PubMed1989

Multihormone regulation of MMTV-LTR in transfected T-47-D human breast cancer cells.

Multihormonal regulation on the long terminal repeat (LTR) region of mouse mammary tumour virus (MMTV) has been studied using T-47-D human breast cancer cells stably transfected with the steroid sensitive LTR-C3 chimaeric gene. The specificity of steroid action on transfected LTR sequences has been compared with regulation of endogenous cellular markers. We conclude that the hormone response element of the LTR can be induced by physiological concentrations of androgen, progestin and glucocorticoid. 17 beta-Oestradiol did not regulate the LTR at physiological levels but an effect was found at 10(-6) M. This effect was not inhibited by antioestrogen nor was it reproduced by the synthetic oestrogen diethylstilboestrol suggesting such effects do not occur via the oestrogen receptor. The antioestrogens tamoxifen and transhydroxytamoxifen do not induce the LTR. No significant steroid competition was found in LTR regulation: whilst oestradiol did not act at physiological concentration it did not interfere with induction by androgen, progestin or glucocorticoid. Such gene regulation did not simply follow receptor status of the cells nor was it reflected in patterns of growth regulation by steroids. The implications of these findings on the mechanism of steroid hormone action are discussed.

Breast Neoplasms↗

PubMed1987

The AT-rich sequence of the SV40 control region influences the binding of SV40 T antigen to binding sites II and III.

During lytic infection SV40 T antigen binds specifically to three different regions of the SV40 DNA to initiate DNA replication and to regulate early and late transcription. We constructed plasmids containing either 23-bp synthetic oligonucleotides representing site I or II or SV40 DNA fragments with combinations of binding sites II and III with or without SV40 specific flanking regions. These plasmids were used to determine which sequences are sufficient for specific binding to isolated regions II and III. Under identical conditions T antigen bound in a sensitive in vitro binding assay efficiently to site I but not to the corresponding oligonucleotide of site II. Binding to site II could only be observed in the presence of the adjacent 17-bp AT-rich region of the SV40 DNA. On account of the markedly low affinity for binding site II, T antigen concentrations were required which exceeded those necessary to achieve saturation of binding to site I. The very low affinity for isolated site III could be slightly raised by the same AT-rich region. An increased binding to site II at 37 degrees compared to 0 degree in the presence of this region points to an indirect influence on the DNA structure of the binding site.

Animals↗

PubMed1988

The enhancer elements and GGGCGG boxes of SV40 provide similar functions in bidirectionally promoting transcription.

The early and the late genes of simian virus 40 (SV40) are transcribed in opposite directions from a shared promoter region. The 72- and the 21-bp repeat regions of the SV40 genome contain the transcriptional enhancer and six copies of the Sp 1-binding GGGCGG box, respectively. SV40 mutants lacking various parts of these regions were examined in COS cells to determine the importance of these sequences for transcription in each direction. We made the following observations. (i) The 72-bp repeat region was required for efficient transcription of both the early and the late genes. (ii) The 21-bp repeat region was required for efficient early-gene transcription, but not for efficient late-gene transcription; however, it was able to supply some late-promoter activity when the 72-bp repeat region was missing. (iii) The ability of either of these regions to promote transcription was gradually reduced as the number of promoter elements within each was decreased. (iv) Mutations in these regions always decreased early-gene transcription more than late-gene transcription. These results indicate that both regions are made up of multiple bidirectional promoter elements, but that the 72-bp repeat region is more effective at inducing transcription than the 21-bp repeat region. Since each region can also (i) satisfy a need for promoter elements in the replication of viral DNA and (ii) induce a region of open chromatin, we conclude that the promoter elements within the enhancer and the GGGCGG boxes probably provide similar functions.

DNA, Viral↗

PubMed1989

A naturally occurring deletion in the enhancer repeats of the human papovavirus BK optimizes early enhancer function at the expense of late promoter activity.

The late promoter of the human papovavirus BK (prototype) is contained within the same region of DNA as the early promoter enhancer. This region, consisting of a 68-50-68-bp repeat and the flanking region to the late side, contains elements important for both late promoter activity and enhancer function for the early transcription unit. We have studied the importance of the naturally occurring 18-bp deletion in the middle repeat by constructing three repeats containing every possible combination of the 50- and 68-bp segments and testing their abilities to promote late transcription and activate an enhancerless SV40 early promoter. We find that the wild-type configuration of repeats gives optimal enhancer function, but is less active than other combinations for late transcription. This suggests that different interactions between the transcription elements contained within the same region of DNA are responsible for modulation of early and late transcription.

BK Virus↗

PubMed1989

Extension of JC virus host range to monkey cells by insertion of a simian virus 40 enhancer into the JC virus regulatory region.

A chimeric polyomavirus genome was constructed by inserting the 72- and the 21-bp repeats of simian virus 40 (SV40) into the JC virus (JCV) regulatory region on the late side of the JCV 98-bp repeats. Although the chimeric polyomavirus DNA was able to replicate very well in human fetal glial cells, deletions were found in sequences of the regulatory region. DNA sequence analysis of a selected clone indicated a 294-bp deletion from the original construction that retained the sequences for the JCV replication origin, 78 bp of one 98-bp repeat, 33 bp of one SV40 72-bp repeat, and one intact 72-bp repeat. Of significant interest was that this genome demonstrated an extended species and cell-type host range, producing infectious virus in human fetal brain and embryonic kidney as well as in rhesus monkey fetal and adult glial cells. However, the species host range was not extended beyond primate cells since the chimeric polyomavirus was unable to multiply in rodent glial cells. Analysis of the viral RNA transcripts from either kidney or glial cells indicated that the major start sites for early RNA mapped within the JCV sequences of the regulatory region and major start sites for late RNA mapped within the JCV and SV40 sequences. This extension of the JC virus host range was most likely attributable to changes in the regulatory region and not the viral T protein since a recombinant DNA clone which placed the coding sequences for the wild-type JCV T protein subject to regulation by the deleted chimeric regulatory region showed a similar extension of host range.

Animals↗

PubMed1989

Polyadenylation function and sequence variability of the long terminal repeats of the human endogenous retrovirus-like family RTVL-H.

The RTVL-H family of human endogenous retrovirus-like sequences consists of approximately 1000 "full-length" elements and at least as many solitary RTVL-H related long terminal repeats (LTRs). We have characterized cDNA clones from two human cell libraries (Hep-2 and normal peripheral blood) and have found three clones in which the AATAAA signal within the RTVL-H LTR has functioned to polyadenylate the transcript. In two of these cases the LTR has provided the polyadenylation signal for non-RTVL-H initiated transcriptional units. The DNA sequences of the LTR regions from these three cDNA clones are significantly different from a consensus LTR sequence generated from 10 genomic LTRs. In fact, two of these cDNA-derived LTRs, although closely related to each other, have a subregion within them which is not found in the genomic LTRs that have been analyzed. LTRs containing this subregion, termed type II LTRs, comprise approximately 25% of the total genomic LTR population. In stable DNA transfection experiments, both a type I and a type II LTR were able to donate a functional polyadenylation signal to a neomycin resistance gene. In LTR-positive placental cDNA clones, type II LTRs were found more frequently than expected from their genomic abundance. These findings suggest that RTVL-H LTRs may provide 3' processing signals for a variety of human RNAs. They also indicate that at least one distinct subpopulation of RTVL-H LTRs can be distinguished and suggest that this or other subpopulations may have different functional capacities in different human cells.

Base Sequence↗

PubMed1990

The role of the direct repeat in qut-controlled antitermination in phage lambda.

For antitermination of transcription from the late p'R promoter of phage lambda, a cis-acting qut sequence, which overlaps with p'R, is required, together with the product of lambda gene Q. Using our BspMI-mediated multicycle technique for generation of precise deletions, we have confirmed that deletions removing DNA downstream of +18 bp (counted from the p'R-controlled transcriptional start point s'R = +1) do not affect the efficiency of qut antitermination; at the same time we found that deleting one more bp (shifting the right-hand boundary to bp +17) reduces antitermination by only 20%. Deleting another 5 or 6 bp (+11 or +12 bp right-hand qut boundary), decreases antitermination by about 80%. These deletions reduce the 9/10-bp-direct repeat (5'-TGGGT(A or T)AATT)2 in qut to only the five italicized bp. Similar strong reduction in antitermination (by about 68%) was obtained with +16 bp qut boundaries, in constructs which also contained 2- or 3-bp insertions between bp +11 and +12 or between +12 and +13. Since the latter deletions retain only 5/10 bp of the direct repeat, it appears that antitermination is dependent on the length and intactness of the direct repeat.

Bacteriophage lambda↗

PubMed1990

Organization and function of the ORIs sequence in the genome of EHV-1 DI particles.

Equine herpesvirus type 1 (EHV-1) cultures enriched for defective interfering particles (DIP) mediate oncogenic transformation and persistent infection in permissive hamster embryo fibroblasts. We have recently demonstrated that an origin of replication (ORI) is located within the central portion (map units 0.828 and 0.948) of the inverted repeat sequence (IRs) of the short region of the standard EHV-1 genome. In the generation of the genome of EHV-1 DI particles, sequences from this internal portion of the IRs recombine with sequences at the long region terminus at nucleotides 3244-3251. In this paper we report that the ORIs sequence is precisely conserved in the DIP genome, that direct repeat sequences near the ORIs sequence which may enhance DNA replication are mutated in the DIP genome, and that the ORI sequence of DIP DNA is functional in DNA replication assays.

Base Sequence↗

PubMed1990

The nucleotide sequence of abutilon mosaic virus reveals prokaryotic as well as eukaryotic features.

The complete nucleotide sequence of abutilon mosaic virus (West Indian isolate, AbMVa) is presented. The resulting genomic structure resembles that of other geminiviruses which are transmitted by the whitefly Bemisia tabaci: AbMV possesses a bipartite circular genome with bidirectional orientation of the open reading frames (ORF). Both components have a common region of 180 bases with 99% homology while the rest of their sequence is distinct. Eukaryotic regulatory transcription elements precede most ORFs and polyadenylation signals are present at the end of most ORFs. However, two ORFs show features of prokaryotic genes. This chimaeric genome organisation is discussed with reference to the finding that AbMV DNA is present in plastids as well as in the nucleus of infected cells.

Amino Acid Sequence↗

PubMed1990

Characterization of the negative regulatory element of the 5' noncoding region of Moloney murine leukemia virus in mouse embryonal carcinoma cells.

An intragenic untranslated region of Moloney murine leukemia virus suppressed gene expression from a long terminal repeat about 30-fold in F9 cells, an embryonal carcinoma (EC) cell line. In another EC line, PCC4 cells and their subline, EA2 cells, and in a fibroblast line, NIH3T3 cells, the suppression by this region was about 4- and 3-fold, respectively. Deletion analysis indicated that the upstream of the previously defined region is required for the suppression. Although not as efficient, this region exhibited suppressive activity even when placed upstream of the transcriptional start site of the long terminal repeat. These results suggest that the suppression is cell type specific and operates partly at the level of the initiation of transcription.

Animals↗

PubMed1991

Regulation of JCVL promoter function: evidence that a pentanucleotide "silencer" repeat sequence AGGGAAGGGA down-regulates transcription of the JC virus late promoter.

The human neurotropic papovavirus JCV contains sequences within the two 98-bp tandem repeats which play a key role in glial-specific transcription of the viral early and late promoters. Previous analysis of the 98-bp sequence has delineated several protein-binding domains that are recognized by nuclear factors present in human brain cells. In the present study, by deletion mutation analysis, we have identified a region within each 98-bp repeat that reduces transcriptional activity of the JCV late promoter (JCVL). Using synthetic oligonucleotides spanning this region, designated "OP," we demonstrate that down-regulation of the JCVL promoter is associated with a pentanucleotide repeat sequence (AGGGAAGGGA) juxtaposed to the poly(dA) tract within the 98-bp tandem repeats. The OP sequence interacts specifically with a protein derived from glial nuclear extract and forms a major 56- to 60-kDa complex. Methylation interference experiment indicates that the three G residues proximal to the poly(dA) tract make major groove contacts with the protein. Single-base-pair substitution of these residues suggests that the complex can form in the presence of two of the three guanosyl residues. The possible role of this protein in regulating the JCV lytic cycle in concert with nearby regulatory elements within JCV promoter region is discussed.

Animals↗