The ARS309 chromosomal replicator of Saccharomyces cerevisiae depends on an exceptional ARS consensus sequence

Autonomously replicating sequence (ARS) elements, which function as the cis-acting chromosomal replicators in the yeast Saccharomyces cerevisiae, depend upon an essential copy of the 11-bp ARS consensus sequence (ACS) for activity. Analysis of the chromosome III replicator ARS309 unexpectedly revealed that its essential ACS differs from the canonical ACS at two positions. One of the changes observed in ARS309 inactivates other ARS elements. This atypical ACS binds the origin recognition complex efficiently and is required for chromosomal replication origin activity. Comparison of the essential ACS of ARS309 with the essential regions of other ARS elements revealed an expanded 17-bp conserved sequence that efficiently predicts the essential core of ARS elements.

IFN consensus sequence binding protein potentiates STAT1-dependent activation of IFNγ-responsive promoters in macrophages

IFNγ, once called the macrophage-activating factor, stimulates many genes in macrophages, ultimately leading to the elicitation of innate immunity. IFNγ's functions depend on the activation of STAT1, which stimulates transcription of IFNγ-inducible genes through the GAS element. The IFN consensus sequence binding protein (icsbγ or IFN regulatory factor 8), encoding a transcription factor of the IFN regulatory factor family, is one of such IFNγ-inducible genes in macrophages. We found that macrophages from ICSBP−/− mice were defective in inducing some IFNγ-responsive genes, even though they were capable of activating STAT1 in response to IFNγ. Accordingly, IFNγ activation of luciferase reporters fused to the GAS element was severely impaired in ICSBP−/− macrophages, but transfection of ICSBP resulted in marked stimulation of these reporters. Consistent with its role in activating IFNγ-responsive promoters, ICSBP stimulated reporter activity in a GAS-specific manner, even in the absence of IFNγ treatment, and in STAT1 negative cells. Indicative of a mechanism for this stimulation, DNA affinity binding assays revealed that endogenous ICSBP was recruited to a multiprotein complex that bound to GAS. These results suggest that ICSBP, when induced by IFNγ through STAT1, in turn generates a second wave of transcription from GAS-containing promoters, thereby contributing to the elicitation of IFNγ's unique activities in immune cells.

Mutations within a furin consensus sequence block proteolytic release of ectodysplasin-A and cause X-linked hypohidrotic ectodermal dysplasia

X-linked hypohidrotic ectodermal dysplasia (XLHED) is a heritable disorder of the ED-1 gene disrupting the morphogenesis of ectodermal structures. The ED-1 gene product, ectodysplasin-A (EDA), is a tumor necrosis factor (TNF) family member and is synthesized as a membrane-anchored precursor protein with the TNF core motif located in the C-terminal domain. The stalk region of EDA contains the sequence -Arg-Val-Arg-Arg156-Asn-Lys-Arg159-, representing overlapping consensus cleavage sites (Arg-X-Lys/Arg-Arg↓) for the proprotein convertase furin. Missense mutations in four of the five basic residues within this sequence account for ≈20% of all known XLHED cases, with mutations occurring most frequently at Arg156, which is shared by the two consensus furin sites. These analyses suggest that cleavage at the furin site(s) in the stalk region is required for the EDA-mediated cell-to-cell signaling that regulates the morphogenesis of ectodermal appendages. Here we show that the 50-kDa EDA parent molecule is cleaved at -Arg156Asn-Lys-Arg159↓- to release the soluble C-terminal fragment containing the TNF core domain. This cleavage appears to be catalyzed by furin, as release of the TNF domain was blocked either by expression of the furin inhibitor α1-PDX or by expression of EDA in furin-deficient LoVo cells. These results demonstrate that mutation of a functional furin cleavage site in a developmental signaling molecule is a basis for human disease (XLHED) and raise the possibility that furin cleavage may regulate the ability of EDA to act as a juxtacrine or paracrine factor.

An AML-1 consensus sequence binds an osteoblast-specific complex and transcriptionally activates the osteocalcin gene.

Tissue and cell-type specific expression of the rat osteocalcin (rOC) gene involves the interplay of multiple transcriptional regulatory factors. In this report we demonstrate that AML-1 (acute myeloid leukemia-1), a DNA-binding protein whose genes are disrupted by chromosomal translocations in several human leukemias, interacts with a sequence essential for enhancing tissue-restricted expression of the rOC gene. Deletion analysis of rOC promoter-chloramphenicol acetyltransferase constructs demonstrates that an AML-1-binding sequence within the proximal promoter (-138 to -130 nt) contributes to 75% of the level of osteocalcin gene expression. The activation potential of the AML-1-binding sequence has been established by overexpressing AML-1 in osteoblastic as well as in nonosseous cell lines. Overexpression not only enhances rOC promoter activity in osteoblasts but also mediates OC promoter activity in a nonosseous human fibroblastic cell line. A probe containing this site forms a sequence specific protein-DNA complex with nuclear extracts from osteoblastic cells but not from nonosseous cells. Antisera supershift experiments indicate the presence of AML-1 and its partner protein core-binding factor beta in this osteoblast-restricted complex. Mutations of the critical AML-1-binding nucleotides abrogate formation of the complex and strongly diminish promoter activity. These results indicate that an AML-1 related protein is functional in cells of the osteoblastic lineage and that the AML-1-binding site is a regulatory element important for osteoblast-specific transcriptional activation of the rOC gene.

The consensus sequence of a major Alu subfamily contains a functional retinoic acid response element.

Alu repeats are interspersed repetitive DNA elements specific to primates that are present in 500,000 to 1 million copies. We show here that an Alu sequence encodes functional binding sites for retinoic acid receptors, which are members of the nuclear receptor family of transcription factors. The consensus sequences for the evolutionarily recent Alu subclasses contain three hexamer half sites, related to the consensus AGGTCA, arranged as direct repeats with a spacing of 2 bp, which is consistent with the binding specificities of retinoic acid receptors. An analysis was made of the DNA binding and transactivation potential of these sites from an Alu sequence that has been previously implicated in the regulation of the keratin K18 gene. These Alu double half sites are shown to bind bacterially synthesized retinoic acid receptors as assayed by electrophoretic mobility shift assays. These sites are further shown to function as a retinoic acid response element in transiently transfected CV-1 cells, increasing transcription of a reporter gene by a factor of approximately 35-fold. This transactivation requires cotransfection with vectors expressing retinoic acid receptors, as well as the presence of all-trans-retinoic acid, which is consistent with the known function of retinoic acid receptors as ligand-inducible transcription factors. The random insertion of potentially thousands of Alu repeats containing retinoic acid response elements throughout the primate genome is likely to have altered the expression of numerous genes, thereby contributing to evolutionary potential.

Chicken interferon consensus sequence-binding protein (ICSBP) and interferon regulatory factor (IRF) 1 genes reveal evolutionary conservation in the IRF gene family.

Members of the IRF family mediate transcriptional responses to interferons (IFNs) and to virus infection. So far, proteins of this family have been studied only among mammalian species. Here we report the isolation of cDNA clones encoding two members of this family from chicken, interferon consensus sequence-binding protein (ICSBP) and IRF-1. The predicted chicken ICSBP and IRF-1 proteins show high levels of sequence similarity to their corresponding human and mouse counterparts. Sequence identities in the putative DNA-binding domains of chicken and human ICSBP and IRF-1 were 97% and 89%, respectively, whereas the C-terminal regions showed identities of 64% and 51%; sequence relationships with mouse ICSBP and IRF-1 are very similar. Chicken ICSBP was found to be expressed in several embryonic tissues, and both chicken IRF-1 and ICSBP were strongly induced in chicken fibroblasts by IFN treatment, supporting the involvement of these factors in IFN-regulated gene expression. The presence of proteins homologous to mammalian IRF family members, together with earlier observations on the occurrence of functionally homologous IFN-responsive elements in chicken and mammalian genes, highlights the conservation of transcriptional mechanisms in the IFN system, a finding that contrasts with the extensive sequence and functional divergence of the IFNs.

Transposon Tc1-derived, sequence-tagged sites in Caenorhabditis elegans as markers for gene mapping

We present an approach to map large numbers of Tc1 transposon insertions in the genome of Caenorhabditis elegans. Strains have been described that contain up to 500 polymorphic Tc1 insertions. From these we have cloned and shotgun sequenced over 2000 Tc1 flanks, resulting in an estimated set of 400 or more distinct Tc1 insertion alleles. Alignment of these sequences revealed a weak Tc1 insertion site consensus sequence that was symmetric around the invariant TA target site and reads CAYATATRTG. The Tc1 flanking sequences were compared with 40 Mbp of a C. elegans genome sequence. We found 151 insertions within the sequenced area, a density of ≈1 Tc1 insertion in every 265 kb. As the rest of the C. elegans genome sequence is obtained, remaining Tc1 alleles will fall into place. These mapped Tc1 insertions can serve two functions: (i) insertions in or near genes can be used to isolate deletion derivatives that have that gene mutated; and (ii) they represent a dense collection of polymorphic sequence-tagged sites. We demonstrate a strategy to use these Tc1 sequence-tagged sites in fine-mapping mutations.

Molecular cloning of a rat chromosome putative recombinogenic sequence homologous to the hepatitis B virus encapsidation signal.

Previously, we reported that a 61-bp subgenomic HBV DNA sequence (designated as 15AB, nt 1855-1915) is a hot spot for genomic recombination and that a cellular protein binding to 15AB may be the putative recombinogenic protein. In the present study, we established the existence of a 15AB-like sequence in human and rat chromosomal DNA by Southern blot analysis. The 15AB-like sequence isolated from the rat chromosome demonstrated a 80.9% identity with 5'-CCAAGCTGTGCCTTGGGTGGC-3', at 1872-1892 of the hepatitis B virus genome, thought to be the essential region for recombination. Interestingly, this 15AB-like sequence also contained the pentanucleotide motifs GCTGG and CCAGC as an inverted repeat, part of the chi known hot spot for recombination in Escherichia coli. Importantly, a portion of the 15AB-like sequence is homologous (82.1%, 23/28 bp) to break point clusters of the human promyelocytic leukemia (PML) gene, characterized by a translocation [t(15;17)], and to rearranged mouse DNA for the immunoglobulin kappa light chain. Moreover, 15AB and 15AB-like sequences have striking homologies (12/15 = 80.0% and 13/15 = 86.7%, respectively) to the consensus sequence for topoisomerase II. Our present results suggest that this 15AB-like sequence in the rat genome might be a recombinogenic candidate triggering genomic instability in carcinogenesis.

Identification, purification, and molecular cloning of autonomously replicating sequence-binding protein 1 from fission yeast Schizosaccharomyces pombe.

Autonomously replicating sequence (ARS) elements of the fission yeast Schizosaccharomyces pombe contain multiple imperfect copies of the consensus sequence reported by Maundrell et al. [Maundrell K., Hutchison, A. & Shall, S. (1988) EMBO J. 7, 2203-2209]. When cell free extracts of S. pombe were incubated with a dimer or tetramer of an oligonucleotide containing the ARS consensus sequence, several complexes were detected using a gel mobility-shift assay. The proteins forming these complexes also bind ars3002, which is the most active origin in the ura4 region of chromosome III of S. pombe. One protein, partly responsible for the binding activity observed with crude extracts, was purified to near homogeneity. It is a 60-kDa protein and was named ARS-binding protein 1 (Abp1). Abp1 preferentially binds to multiple sites in ARS 3002 and to the DNA polymer poly[d(A.T)]. The cloning and sequence of the gene coding for Abp1 revealed that it encodes a protein of 59.8 kDa (522 amino acids). Abp1 has significant homology (25% identity, 50% similarity) to the N-terminal region (approximately 300 amino acids) of the human and mouse centromere DNA-binding protein CENP-B. Because centromeres of S. pombe contain a high density of ARS elements, Abp1 may play a role connecting DNA replication and chromosome segregation.

Impaired fetal T cell development and perinatal lethality in mice lacking the cAMP response element binding protein

CREB, the cAMP response element binding protein, is a key transcriptional regulator of a large number of genes containing a CRE consensus sequence in their upstream regulatory regions. Mice with a hypomorphic allele of CREB that leads to a loss of the CREBα and Δ isoforms and to an overexpression of the CREBβ isoform are viable. Herein we report the generation of CREB null mice, which have all functional isoforms (CREBα, β, and Δ) inactivated. In contrast to the CREBαΔ mice, CREB null mice are smaller than their littermates and die immediately after birth from respiratory distress. In brain, a strong reduction in the corpus callosum and the anterior commissures is observed. Furthermore, CREB null mice have an impaired fetal T cell development of the αβ lineage, which is not affected in CREBαΔ mice on embryonic day 18.5. Overall thymic cellularity in CREB null mice is severely reduced affecting all developmental stages of the αβ T cell lineage. In contrast γδ T cell differentiation is normal in CREB mutant mice.

A cobalt complex that selectively disrupts the structure and function of zinc fingers

Zinc finger domains are structures that mediate sequence recognition for a large number of DNA-binding proteins. These domains consist of sequences of amino acids containing cysteine and histidine residues tetrahedrally coordinated to a zinc ion. In this report, we present a means to selectively inhibit a zinc finger transcription factor with cobalt(III) Schiff-base complexes. 1H NMR spectroscopy confirmed that the structure of a zinc finger peptide is disrupted by axial ligation of the cobalt(III) complex to the nitrogen of the imidazole ring of a histidine residue. Fluorescence studies reveal that the zinc ion is displaced from the model zinc finger peptide in the presence of the cobalt complex. In addition, gel-shift and filter-binding assays reveal that cobalt complexes inhibit binding of a complete zinc finger protein, human transcription factor Sp1, to its consensus sequence. Finally, a DNA-coupled conjugate of the cobalt complexes selectively inhibited Sp1 in the presence of several other transcription factors.

Identification of thyroid hormone response elements in the human fatty acid synthase promoter

To investigate the regulation of the human fatty acid synthase gene by the thyroid hormone triiodothyronine, various constructs of the human fatty acid synthase promoter and the luciferase reporter gene were transfected in combination with plasmids expressing the thyroid hormone and the retinoid X receptors in HepG2 cells. The reporter gene was activated 25-fold by the thyroid hormone in the presence of the thyroid hormone receptor. When both the thyroid hormone and the retinoid X receptors were expressed in HepG2 cells, there was about a 100-fold increase in reporter gene expression. 5′-Deletion analysis disclosed two thyroid hormone response elements, TRE1 (nucleotides −870 to −650) and TRE2 (nucleotides −272 to −40), in the human fatty acid synthase promoter. The presence of thyroid hormone response elements in these two regions of the promoter was confirmed by cloning various fragments of these two regions in the minimal thymidine kinase promoter−luciferase reporter gene plasmid construct and determining reporter gene expression. The results of this cloning procedure and those of electrophoretic mobility shift assays indicated that the sequence GGGTTAcgtcCGGTCA (nucleotides −716 to −731) represents TRE1 and that the sequence GGGTCC (nucleotides −117 to −112) represents TRE2. The sequence of TRE1 is very similar to the consensus sequence of the thyroid hormone response element, whereas the sequence of TRE2 contains only a half-site of the thyroid hormone response element consensus motif because it lacks the direct repeat. The sequences on either side of TRE2 seem to influence its response to the thyroid hormone and retinoid X receptors.

Purification and characterization of a CENP-B homologue protein that binds to the centromeric K-type repeat DNA of Schizosaccharomyces pombe

We have purified and characterized a novel 60-kDa protein that binds to centromeric K-type repeat DNA from Schizosaccharomyces pombe. This protein was initially purified by its ability to bind to the autonomously replicating sequence 3002 DNA. Cloning of the gene encoding this protein revealed that it possesses significant homology to the mammalian centromere DNA-binding protein CENP-B and S. pombe Abp1, and this gene was designated as cbh+ (CENP-B homologue). Cbh protein specifically interacts in vitro with the K-type repeat DNA, which is essential for centromere function. The Cbh-binding consensus sequence was determined by DNase I footprinting assays as PyPuATATPyPuTA, featuring an inverted repeat of the first four nucleotides. Based on its binding activity to centromeric DNA and homology to centromere proteins, we suggest that this protein may be a functional homologue of the mammalian CENP-B in S. pombe.

Transcripts from a single full-length cDNA clone of hepatitis C virus are infectious when directly transfected into the liver of a chimpanzee

We have succeeded in constructing a stable full-length cDNA clone of strain H77 (genotype 1a) of hepatitis C virus (HCV). We devised a cassette vector with fixed 5′ and 3′ termini and constructed multiple full-length cDNA clones of H77 in a single step by cloning of the entire ORF, which was amplified by long reverse transcriptase–PCR, directly into this vector. The infectivity of two complete full-length cDNA clones was tested by the direct intrahepatic injection of a chimpanzee with RNA transcripts. However, we found no evidence for HCV replication. Sequence analysis of these and 16 additional full-length clones revealed that seven clones were defective for polyprotein synthesis, and the remaining nine clones had 6–28 amino acid mutations in the predicted polyprotein compared with the consensus sequence of H77. Next, we constructed a consensus chimera from four of the full-length cDNA clones with just two ligation steps. Injection of RNA transcripts from this consensus clone into the liver of a chimpanzee resulted in viral replication. The sequence of the virus recovered from the chimpanzee was identical to that of the injected RNA transcripts. This stable infectious molecular clone should be an important tool for developing a better understanding of the molecular biology and pathogenesis of HCV.

Attenuation of virulence in Mycobacterium tuberculosis expressing a constitutively active iron repressor

Iron is an essential nutrient for the survival of most organisms and has played a central role in the virulence of many infectious disease pathogens. Mycobacterial IdeR is an iron-dependent repressor that shows 80% identity in the functional domains with its corynebacterial homologue, DtxR (diphtheria toxin repressor). We have transformed Mycobacterium tuberculosis with a vector expressing an iron-independent, positive dominant, corynebacterial dtxR hyperrepressor, DtxR(E175K). Western blots of whole-cell lysates of M. tuberculosis expressing the dtxR(E175K) gene revealed the stable expression of the mutant protein in mycobacteria. BALB/c mice were infected by tail vein injection with 2 × 105 organisms of wild type or M. tuberculosis transformed with the dtxR mutant. At 16 weeks, there was a 1.2 log reduction in bacterial survivors in both spleen (P = 0.0002) and lungs (P = 0.006) with M. tuberculosis DtxR(E175K). A phenotypic difference in colonial morphology between the two strains also was noted. A computerized search of the M. tuberculosis genome for the palindromic consensus sequence to which DtxR and IdeR bind revealed six putative “iron boxes” within 200 bp of an ORF. Using a gel-shift assay we showed that purified DtxR binds to the operator region of five of these boxes. Attenuation of M. tuberculosis can be achieved by the insertion of a plasmid containing a constitutively active, iron-insensitive repressor, DtxR(E175K), which is a homologue of IdeR. Our results strongly suggest that IdeR controls genes essential for virulence in M. tuberculosis.