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PubMed1999

Ethnic variation in the thymidylate synthase enhancer region polymorphism among Caucasian and Asian populations.

Thymidylate synthase (TS) regulates the production of DNA synthesis precursors and is an important target of cancer chemotherapy. A tandem repeat sequence in a TS promoter enhancer region (TSER) was recently identified. Polymorphic variation affected in vitro expression levels of the gene. We evaluated the influence of ethnicity on TSER genotype. Allele frequency was similar in Caucasian and Southwest Asian subjects. However, homozygous triple repeat subjects were twice as common in Chinese subjects (67%) than in Caucasian subjects (38%). This demonstrates significant ethnic variation in a TS gene regulatory element which may have significant impact on pyrimidine homeostasis and drug therapy.

Alleles↗
PubMed2000

Sequence analyses of the olfactory receptor gene cluster mOR37 on mouse chromosome 4.

The olfactory receptor multigene family is organized in clusters spread throughout the genome. In the present study, we have sequenced two subregions of the mOR37 gene cluster on mouse chromosome 4. The resulting 100 kb of sequence revealed seven odorant receptor coding regions and one gene fragment. Sequence analyses reveal that the mOR37 gene cluster may represent a rather ancient cluster. The mOR37 genes exhibit a complex intron/exon structure, and some appear to be differentially spliced. All genes in the cluster share conserved sequence motifs 5' of their putative initial exons, which represent potential binding sites for transcription factors. The clustered organization and conserved sequence motifs suggest common expression control mechanisms for these genes.

5' Untranslated Regions↗
PubMed1993

Multiple transitions to non-B-DNA structures occur in the distal regulatory region of the rat prolactin gene.

The developmentally regulated rat prolactin (rPRL) gene presents a promising model system toward understanding the biological role of non-B-DNA structural elements. Two predominantly alternating purine-pyrimidine (APP) (dA-dC)n.(dG-dT)n repeats of 58 and 178 base-pairs flank the (A + T)-rich distal regulatory region. We have characterized several transitions to non-B-DNA structures within this region in negatively supercoiled plasmids by utilizing high resolution chemical probing. Each repeat undergoes a full-length conversion to a novel left-handed helical structure via the stepwise nucleation and propagation of discrete "segments". These segments are delimited by out-of-alternation bases that are susceptible to attack by potassium permanganate and thus appear to be significantly unstacked within the left-handed helices. Moreover, the spatial order of successive right- to left-handed DNA transitions within each repeat exhibits a clear polarity toward the distal regulatory region of the rPRL gene. An additional transition involving the long-range unpairing of (A + T)-rich sequences establishes a directional propagation toward the regulatory region. These data demonstrate a complex series of quasi-independent transitions to non-B-DNA structures that impinge upon a known regulatory control region.

Animals↗
PubMed1993

Two secretory protein genes in Chironomus tentans have arisen by gene duplication and exhibit different developmental expression patterns.

The salivary gland cells in the dipteran Chironomus tentans produce approximately 15 different secretory proteins, with relative molecular masses ranging between 1 x 10(4) and 1 x 10(6). Together these proteins form two types of extra corporal tubes, a larval protective housing and feeding tube or a pupation tube. The developmental change in tube formation is accompanied by a switch in production from one combination of secretory proteins to another. Here we characterize two genes, the sp38-40.A and B genes, which encode secretory proteins with relative molecular masses of 38,000 to 40,000. The two genes are located 346 base-pairs apart in the same orientation and have presumably arisen by gene duplication as the result of an illegitimate recombination event. Both genes contain two regions with cysteine codons, surrounded by regions with short repeats coding for proline and charged amino acid residues. The two genes and alleles of the genes differ in their number of repeats. This structure resembles the structure of the Balbiani ring (BR) genes, which encode the four largest salivary gland secretory proteins. The sp38-40.A and B genes are therefore likely to belong to a BR multigene family containing all or most of the 15 salivary gland secretory protein genes. The expression of the sp38-40.A and B genes are different: the A gene is expressed throughout the larval fourth instar but considerably less in the prepupal stage, while the B gene shows the opposite expression pattern. The developmental regulation of the expression of the two genes has therefore diverged after the gene duplication event.

Amino Acid Sequence↗
PubMed2000

Functional characterization of the enhancer blocking element of the sea urchin early histone gene cluster reveals insulator properties and three essential cis-acting sequences.

Insulator elements can be functionally identified by their ability to shield promoters from regulators in a position-dependent manner or their ability to protect adjacent transgenes from position effects. We have previously reported the identification of a 265 bp sns DNA fragment at the 3' end of the sea urchin H2A early histone gene that blocked expression of a reporter gene in transgenic embryos when placed between the enhancer and the promoter. Here we show that sns interferes with enhancer-promoter interaction in a directional manner. When sns is placed between the H2A modulator and the inducible tet operator, the modulator is barred from interaction with the basal promoter. However, the tet activator (tTA) can still activate the promoter, even in the presence of sns, demonstrating that sns does not interfere with activity of a downstream enhancer. In addition, the H2A modulator can still drive expression of a divergently oriented transcription unit, suggesting that sns does not inhibit binding of transcription factor(s) to the enhancer. To identify cis-acting sequence elements within sns which are responsible for insulator activity, we have performed in vitro DNase I footprinting and EMSA analysis, and in vivo functional assays by microinjection into sea urchin embryos. We have identified three binding sites for protein complexes: a palindrome, a direct repeat, and a C+T sequence that corresponds to seven GAGA motifs on the transcribed strand. Insulator function requires all three cis-acting elements. Based on these results, we conclude that sns displays properties similar to the best characterized insulators and suggest that directional blocking of enhancer-activated transcription by sns depends on the assembly of distinct DNA-protein complexes.

Animals↗
PubMed2002

A novel TCTG(G/C) direct repeat and an A/T-rich HMG2-binding site control the expression of the rat cardiac troponin T gene.

We previously showed that the rat cardiac troponin T proximal promoter (-497bp from the transcriptional start site) was sufficient to confer cardiac-specific expression of a reporter gene in both cultured cardiomyocytes and transgenic mice. This promoter consists of two cis-regulatory modules with high sequence similarity. Nucleotides from -319 to -289 (termed D2) and nucleotides from -249 to -209 (termed F41) contain a TCTG(G/C) direct repeat and an A/T-rich sequence. Competition gel mobility shift assays revealed that the same protein factors were bound to both D2 and F41. Deletion and religation studies of the promoter suggested that D2 acted as an enhancer but could not totally substitute for the F41 function. Mutational analyses demonstrated that the direct repeat was required for the DNA-binding and promoter activity. Moreover, cardiac-specific 42kDa proteins and a ubiquitous high mobility group 2 (HMG2) protein were identified to be responsible for the binding to the TCTG(G/C) direct repeat and the A/T-rich sequence, respectively. Overexpression of HMG2 had differential effects on the promoter in cardiomyocytes versus fibroblasts. Our results provided the first evidence to support that HMG2 together with tissue-specific factors could direct a stimulatory or inhibitory effect on thecardiac troponin T gene expression in heart or non-heart tissue, respectively.

AT Rich Sequence↗
PubMed2000

Deletion mutagenesis of the 5' psbA2 region in Synechocystis 6803: identification of a putative cis element involved in photoregulation.

Three Synechocystis 6803 mutants, with deletions in the upstream region of the light-regulated psbA2 gene, were constructed. All three mutants grew photoautotrophically but they were affected in psbA2 expression. In one of the mutants, DeltaU80, psbA2 transcription levels were not suppressed under low light conditions and production of psbA2 transcripts occurred also in darkness, in contrast to the situation in the control strain. Comparative sequence analyses of the mutants identified a hexanucleotide repeat as possible cis element in psbA2 light regulation. Electrophoresis mobility shift assays showed that protein binding to the hexanucleotide repeat decreased with light intensity, consistent with a hypothesis that the repeat harbors a binding site for a repressor protein. Repeats similar to that in psbA2 were found also in the upstream region of other light-regulated genes in Synechocystsis 6803. We refer to these hexanucleotide repeats as High Light Regulatory 1 (HLR1) sequences.

Base Sequence↗
PubMed1993

Regulatory elements involved in tax-mediated transactivation of the HTLV-I LTR.

HTLV-I is the etiologic agent of adult T-cell leukemia. In this study, we investigated the regulatory elements and cellular transcription factors which function in modulating HTLV-I gene expression in response to the viral transactivator protein, tax. Transfection experiments into Jurkat cells of a variety of site-directed mutants in the HTLV-1 LTR indicated that each of the three motifs A, B, and C within the 21-bp repeats, the binding sites for the Ets family of proteins, and the TATA box all influenced the degree of tax-mediated activation. Tax is also able to activate gene expression of other viral and cellular promoters. Tax activation of the IL-2 receptor and the HIV-1 LTR is mediated through NF-kappa B motifs. Interestingly, sequences in the 21-bp repeat B and C motifs contain significant homology with NF-kappa B regulatory elements. We demonstrated that an NF-kappa B binding protein, PRDII-BF1, but not the rel protein, bound to the B and C motifs in the 21-bp repeat. PRDII-BF1 was also able to stimulate activation of HTLV-I gene expression by tax. The role of the Ets proteins on modulating tax activation was also studied. Ets 1 but not Ets 2 was capable of increasing the degree of tax activation of the HTLV-I LTR. These results suggest that tax activates gene expression by either direct or indirect interaction with several cellular transcription factors that bind to the HTLV-I LTR.

Base Sequence↗
PubMed1994

Second-site mutations in the brome mosaic virus RNA3 intercistronic region partially suppress a defect in coat protein mRNA transcription.

An intercistronic oligo(A) tract is present in the genomic RNA3 of all bromoviruses sequenced to date and, for brome mosaic virus (BMV), is known to function as an activating sequence in transcription of the subgenomic coat protein mRNA, RNA4. Mutations able to partially compensate for removal of the oligo(A) from BMV RNA3 were identified by obtaining spontaneous second-site revertants. The starting BMV RNA3 mutant carried a deletion of 17 of 18 residues of the intercistronic oligo(A), resulting in a nearly complete loss of subgenomic coat protein mRNA synthesis and reduced RNA3 accumulation. The responsible suppressor mutations acted in cis and were located in the 244-base RNA3 intercistronic region containing the original deletion. Three mutations associated with the revertant phenotype were characterized: (i) A single U-->A substitution in the core subgenomic mRNA promoter restored 35% of wild-type promoter activity. (ii) A duplication of 8 bases (UAUUAUUA) immediately 5' to the oligo(A) deletion site resulted in higher levels of both RNA3 and RNA4 accumulation. (iii) A point substitution in a conserved cellular motif corresponding to box B of RNA polymerase III promoters reduced RNA3 accumulation. Together with certain intervirally conserved promoter sequences, the spontaneous adaptation of a bromovirus subgenomic promoter to function without its unusual oligo(A) activator suggests that bromovirus subgenomic mRNA transcription may share underlying mechanistic similarities with the many other members of the alphavirus-like superfamily, whose subgenomic promoters all lack an oligo(A) tract.

Base Sequence↗
PubMed1995

Aura virus is a New World representative of Sindbis-like viruses.

Aura virus is an alphavirus present in Brazil and Argentina that is serologically related to Sindbis virus (present throughout the Old World) and to Western equine encephalitis (WEE) virus (present in the Americas). We have previously shown that WEE is a recombinant virus whose glycoproteins and part of whose 3' nontranslated region (NTR) are derived from a Sindbis-like virus, but the remainder of whose genome is derived from Eastern equine encephalitis (EEE) virus. We show here that Aura virus is a Sindbis-like virus that shares considerable organizational and sequence identity with Sindbis virus. Certain nucleotide sequence elements present in Aura RNA that are believed to function as promoters are almost identical to their Sindbis counterparts, repeated elements in the 3' nontranslated region are shared with Sindbis virus, and important antigenic epitopes are conserved between the two viruses. Despite their close relationship, the two viruses have diverged significantly, sharing 73% amino acid sequence identity in the nonstructural proteins and 62% identity in the structural proteins. This is about the same as the identities between EEE and Venezuelan equine encephalitis virus, whose promoter elements, 3' NTRs, and antigenic epitopes have diverged more radically, such that these two viruses are considered to belong to different subgroups. Importantly, the glycoproteins of WEE are more closely related to those of Sindbis than to those of Aura virus. From this we propose that an ancestral Sindbis-like virus present in the Americas (probably South America) diverged 1000-2000 years ago into a lineage that gave rise to Aura virus and a lineage that gave rise to Sindbis virus and to the Sindbis-like parent of WEE. At some time after this divergence, a Sindbis-like virus belonging to the latter lineage was transferred to the Old World where it gave rise to Sindbis viruses distributed throughout the Old World, and in a separate event a Sindbis-like virus belonging to the same lineage underwent recombination with EEE to give rise to WEE.

Alphavirus↗
PubMed1995

cis-acting regulatory elements in the bovine immunodeficiency virus long terminal repeat.

Functional cis-acting regulatory elements in the bovine immunodeficiency virus (BIV) long terminal repeat (LTR) were identified by deletion mapping and nuclear protein gel shift analysis using three BIV-infectible cell lines, Cf2Th, BLAC-20, and EREp. Deletion mapping studies indicated that putative NF-kappa B, GRE, AP-4, AP-1, CAAT, and ATF/CRE transcription factor elements positively contribute to LTR-directed gene expression in each cell line both in the presence and absence of the viral transactivator Tat. Sp1 and overlapping AP-3 and retroviral core enhancer elements had variable effects on LTR-directed gene expression depending on cell type and presence or absence of Tat. In addition, a sequence spanning the LTR U5 region and the untranslated viral leader was strongly repressive in all cell lines. Tat transactivated the LTR 25-fold over basal levels in a TAR-dependent manner in Cf2Th cells. In contrast, Tat transactivated the LTR only 2.5-fold over basal levels in EREp and BLAC-20 cells in a TAR-independent manner. Probes for putative NF-kappa B, GRE, Sp1, AP-4, AP-1, overlapping AP-3 and retroviral core enhancer, and juxtaposed CAAT and ATF-CRE elements specifically bound nuclear proteins from these three cell lines and HeLa cells, with the stoichiometry of binding being cell-type dependent. Probes for AP-4, AP-1, and juxtaposed CAAT and ATF/CRE elements exhibited greater protein binding with extracts from virally infected cells than with extracts from uninfected cells, suggesting that viral infection can modulate nuclear factor binding. The present studies indicate that several transcription factor elements in the BIV LTR have functional roles and that cell type can strongly determine the role they play in gene expression.

Animals↗
PubMed1996

Human JC virus cAMP response elements functional for enhanced glial cell expression in differentiating embryonal carcinoma cells.

Human JC virus (JCV) is glial cell-specific for growth and expression. This specificity is attributed to the cis-acting sequences of the two 98-bp tandem repeats in the JCV regulatory region. JCV causes brain lesions, especially in AIDS patients. To study the expression of JCV in glial cells, the role of both repeat region TGAGCTCA sequences, which are homologous to the classical TGAGCTCA cAMP response element (CRE), was examined. The effect of the CRE on expression of the JCV early promoter (JCVE) in response to cAMP was studied with undifferentiated, glial and muscle P19 embryonal carcinoma cells. The results showed a threefold increase in response to cAMP only in the glial cells in which JCV is efficiently expressed. The direct in vivo role of the JCV CRE was confirmed by site-directed mutagenesis. Additionally, a CRE oligonucleotide was induced by cAMP in vivo, and in in vitro transcription assays with glial cell extracts. The early promoter of human BK virus containing nonhomologous CRE sequences was previously shown not to be glial cell-specific and failed to respond to cAMP in glial P19 cells in this study. Mobility shift assays showed the cAMP-induced in vitro interaction of glial cell protein(s) with the CRE oligonucleotide. Southwestern blot and uv crosslinking experiments identified an approximately 43-kDa protein interacting with the JCV CRE oligonucleotide. The results indicate that the in vivo expression of JCVE is specifically increased in response to cAMP only in glial cells and JCV CRE in vitro protein complexes are only detected in response to cAMP for glial cell extracts.

Animals↗
PubMed1996

Activation of the HTLV-I long terminal repeat by the hepatitis B virus X protein.

The human T-cell leukemia virus type I (HTLV-I) Tax protein and the hepatitis B virus (HBV) X protein have each been shown to activate transcription of their respective viral promoters as well as a subset of cellular gene promoters. Here we show that the HTLV-I long terminal repeat (LTR) is responsive to HBV X transactivation. Maximum levels of X-mediated transactivation of the LTR were 8-fold. An X-responsive-region (XRR) of the LTR is located between nucleotides -355 and -276 and contains an AP-2 binding site, a previously recognized X-responsive element. We demonstrated that Tax and X synergize to activate transcription from the HTLV-I LTR, although the AP-2 binding site was not required for this synergy. These results raise the possibility that the HBV X protein may affect the level of HTLV-I gene expression in co-infected individuals.

Base Sequence↗
PubMed1996

The effect of regulatory sequence elements upon the initiation of DNA replication of the minute virus of mice.

The minute virus of mice (MVM) genome is a linear single-stranded length of approximately 5000 nucleotides of DNA with unique terminal palindromic sequences at both ends. The left(3') hairpin is used to prime the initiation of DNA synthesis on parental single-strand DNA while the right (5') hairpin or stem-plus-arms structure can also prime the initiation of DNA synthesis during synthesis of dimer and higher oligomers as well as synthesis of progeny single strands. Previous studies have shown that if viral duplex DNA was input into an in vitro DNA replication system using extracts from uninfected HeLa cells, the 5' end of the molecule was able to form a hairpin and initiate DNA synthesis by DNA polymerase delta (Cossons et al. (1996), Virology 216, 258-264). In this study, the effect of the deletion of known cis-acting genetic elements upon the initiation of DNA replication was studied using a series of MVM mutants with deletions within the 5' terminal region. Mutants containing deletions of elements A (nucleotides 4489-4636), B (nucleotides 4636-4695), and either one or both of the 65-bp repeats (nucleotides 4720-4785 and 4785-4849) were used as template in the in vitro DNA replication system. When element A was deleted, the efficiency of initiation decreased significantly. Subsequent removal of element B, leaving just the two 65-bp repeats, restored levels of initiation back to those seen in the wild-type genome. In the absence of either A or B both 65-bp repeats were necessary for efficient initiation, and removal of one of these repeats caused a decrease in efficiency. Thus, element B appeared to have a negative regulatory effect (in the absence of element A), and element A appeared to have a positive regulatory effect, at least in the presence of element B. These data demonstrate, for the first time, a complex interaction between these cis-acting regulatory elements which can function as both positive or negative regulators in the initiation of MVM DNA replication.

Animals↗
PubMed1997

Comparison of the role of 5' terminal sequences of alfalfa mosaic virus RNAs 1, 2, and 3 in viral RNA replication.

The 5' untranslated regions (UTRs) of the genomic RNAs 1, 2, and 3 of alfalfa mosaic virus (AMV) are 100, 54, and 345 nucleotides (nt) long, respectively, and lack extensive sequence similarity to each other. RNA 3 encodes the movement protein P3 and the coat protein and can be replicated in transgenic tobacco plants expressing the replicase proteins P1 and P2 (P12 plants). 5' Cis-acting sequences involved in RNA 3 replication have been shown to be confined to the 5' UTR. When the 5' UTR of RNA 3 was replaced by the 5' UTRs of RNAs 1 or 2, the recombinant RNA was not infectious to P12 plants. Also, when the P3 gene in RNA 3 was put under the control of a subgenomic promoter and the 5' UTR of this RNA was replaced by 5' terminal RNA 1 sequences of 103 to 860 nt long or RNA 2 sequences of 57 to 612 nt long, no accumulation of the hybrid RNAs was observed. Deletion of the 5' 22 nucleotides of RNA 3 resulted in the accumulation of a major progeny that lacked the 5' 79 nt. However, when the 5' 22 nucleotides of RNA 3 were replaced by the complete 5' UTR of RNA 1 or 5' sequences of RNAs 1, 2, or 3 with a length of 5 to 15 nt, accumulation of the full-length mutant RNAs was observed. The effect of mutations in the 5' viral sequences of 5 to 15 nt was analyzed. It is concluded that although elements within nucleotides 80-345 of the 5' UTR of RNA 3 are sufficient for replication, a specific sequence of 3 to 5 nt is required to target the replicase to an initiation site corresponding to the 5' end of the RNA.

Alfalfa mosaic virus↗
PubMed1994

Tissue-specific and temporal regulation of a beta-conglycinin gene: roles of the RY repeat and other cis-acting elements.

Upstream regulatory sequences (URS) of the gene that encodes the alpha' subunit of beta-conglycinin, the 7S soybean seed storage protein, includes two RY repeat elements. The role of RY elements and sequences that bind soybean embryo factors 3 and 4 (SEF3 and SEF4; Allen et al., Plant Cell 1 (1989) 623-631) in regulating expression of the promoter was studied following site directed mutagenesis. Specific mutations introduced into these sequences abolished the in vitro binding activities of SEF3 and SEF4. The biological activities resulting from the mutations were determined in transgenic plants using two chimeric promoters comprising sequences from the CaMV 35S promoter and the alpha' subunit promoter. The uidA reporter gene was used to assess the levels of gene expression in transgenic plants. The mutations in the RY element and SEF3 and SEF4 binding sites had little effect on expression of the alpha' promoter. By contrast, mutations in the RY element had significant effect on gene expression when the URS from the alpha' promoter was ligated upstream of the core 35S promoter. Mutations in the RY element abolished the seed specific enhancing activity of the alpha' URS and caused expression of the chimeric promoter in leaves. These results indicate that the RY element plays a key role in seed-specific gene regulation in coordination with other cis-acting elements.

Antigens, Plant↗