Tissue specificity of a glucocorticoid-dependent enhancer in transgenic mice.

The glucocorticoid-responsive units (GRUs) of the rat tyrosine aminotransferase were associated with the regulatory sequences of a cellular gene expressed ubiquitously--that coding for the largest subunit of RNA polymerase II. In transient expression assays, glucocorticoid responsiveness of the hybrid regulatory regions depends on the spatial relationship and number of regulatory elements. Two parameters affect the ratio of induction by glucocorticoids: the basal level of the hybrid promoter that is affected by the RNA polymerase II regulatory sequences and the glucocorticoid-induced level that depends on the distance between the GRUs and the TATA box. A fully active glucocorticoid-responsive hybrid gene was used to generate transgenic mice. Results show that a composite regulatory pattern is obtained: ubiquitous basal expression characteristic of the RNA polymerase II gene and liver-specific glucocorticoid activation characteristic of the tyrosine aminotransferase GRUs. This result demonstrates that the activity of the tyrosine aminotransferase GRUs is cell-type-specific not only in cultured cells but also in the whole animal.

Tissue Culture-Specific Expression of a Naturally Occurring Tobacco Feedback-Insensitive Anthranilate Synthase1

A cDNA and corresponding promoter region for a naturally occurring, feedback-insensitive anthranilate synthase (AS) α-subunit gene, ASA2, has been isolated from an unselected, but 5-methyl-tryptophan-resistant (5MTr), tobacco (Nicotiana tabacum) cell line (AB15–12-1). The ASA2 cDNA contains a putative transit peptide sequence, and Southern hybridization shows that more than one closely related sequence is present in the tobacco genome. The ASA2 cDNA complemented a trpE nonsense mutant Escherichia coli strain, allowing growth on 300 μm 5MT-containing minimal medium without tryptophan, and cell extracts contained feedback-insensitive AS activity. The 5MTr was lost when the E. coli strain was transformed with an ASA2 site-directed mutant (phenylalanine-107-arginine-108 → serine-107-glutamine-108). Identical nucleotide sequences encoding the phenylalanine-107-arginine-108 region have been found in polymerase chain reaction-amplified 326-bp ASA2 genomic fragments of wild-type (5-methyl-tryptophan-sensitive [5MTs]) tobacco and a progenitor species. High-level ASA2 transcriptional expression was detected only in 5MTr-cultured cells, not in 5MTs cells or in plants. Promoter studies indicate that tissue specificity of ASA2 is controlled by the promoter region between −2252 and −607. Since the ASA2 promoter sequences are not substantially different in the 5MTr and 5MTs lines, the increased levels of ASA2 mRNA in the 5MTr lines are most likely due to changes in a regulatory gene affecting ASA2 expression.

Combinations of closely situated cis-acting elements determine tissue-specific patterns and anterior extent of early Hoxc8 expression.

We have used a transgene mutation approach to study how expression domains of Hoxc8 are established during mouse embryogenesis. A cis-regulatory region located 3 kb upstream from the Hoxc8 translational start site directs the early phase of expression. Four elements, termed A, B, C, and D, were previously shown to direct expression to the neural tube. Here we report that a fifth element, E, located immediately downstream of D directs expression to mesoderm in combination with the other four elements. These elements are interdependent and partially redundant. Different combinations of elements determine expression in different posterior regions of the embryo. Neural tube expression is determined minimally by ABC, ABD, or ACD; somite expression by ACDE; and lateral plate mesoderm expression by DE. Neural tube and lateral plate mesoderm enhancers can be separated, but independent somite expression has not been achieved. Furthermore, mutations within these elements result in posteriorization of the reporter gene expression. Thus, the anterior extent of expression is determined by the combined action of these elements. We propose that the early phase of Hoxc8 expression is directed by two separate mechanisms: one that determines tissue specificity and another that determines anterior extent of expression.

Identification of genes required for Drosophila eye development using a phenotypic enhancer-trap

A novel method of P-element mutagenesis is described for the isolation of mutants affecting the development of the Drosophila compound eye. It exploits the interaction between the Bride of Sevenless (Boss) ligand and the Sevenless (Sev) receptor tyrosine kinase that triggers the formation of the UV-sensitive photoreceptor neuron, R7. Transposition of a boss cDNA transgene, in an otherwise boss mutant background, was used as a “phenotypic trap” in live flies to identify enhancers expressed during a narrow time window in eye development. Using a rapid behavioral screen, more than 400,000 flies were tested for restoration of R7. Some 1,800 R7-containing flies were identified. Among these, 21 independent insertions with expression of the boss reporter gene in the R8 cell were identified by a external eye morphology and staining with an antibody against Boss. Among 900 lines with expression of the boss reporter gene in multiple cells assessed for homozygous mutant phenotypes, insertions in the marbles, glass, gap1, and fasciclin II genes were isolated. This phenotypic enhancer-trap facilitates (i) the isolation of enhancer-traps with a specific expression pattern, and (ii) the recovery of mutants disrupting development of specific tissues. Because the temporal and tissue specificity of the phenotypic trap is dependent on the choice of the marker used, this approach can be extended to other tissues and developmental stages.

Genes regulated by androgen in the rat ventral prostate

Genes that are regulated by androgen in the prostate were studied in the rat. Four of the less than 10 genes that are down-regulated by androgen in the ventral prostate of a 7-day castrated rat were identified; their mRNAs decayed with identical kinetics. Twenty-five of the estimated 56 genes that are up-regulated by androgen in the castrated prostate have been isolated. The up-regulated genes fall into two kinetic types. Early genes are significantly up-regulated by 6.5 hr whereas the delayed genes respond mainly after 24 hr from the time of androgen replacement. These androgen-response genes are also regulated in the prostate by castration, indicating that these genes could play important roles in androgen-induced regrowth and/or castration-induced regression of the prostate during hormonal manipulation. A survey of the tissue specificity showed that the androgen-response gene expression program in the prostate is mainly prostate-specific. Total RNA Northern blot analysis detects the expression of about 16 up-regulated genes and 3 down-regulated genes in the prostate only. Four up-regulated genes and one down-regulated gene are regulated by androgen in both the prostate and seminal vesicles but not in other organs. The expression of the remaining androgen-response genes is not limited to the prostate but is only responsive to androgen in the prostate. This survey of the androgen-response gene expression program provides insights into the molecular and cellular mechanisms of androgen action in the prostate.

Collagenase-3 Induction in Rat Lung Fibroblasts Requires the Combined Effects of Tumor Necrosis Factor-α and 12-Lipoxygenase Metabolites: A Model of Macrophage-induced, Fibroblast-driven Extracellular Matrix Remodeling during Inflammatory Lung Injury

The mechanisms responsible for the induction of matrix-degrading proteases during lung injury are ill defined. Macrophage-derived mediators are believed to play a role in regulating synthesis and turnover of extracellular matrix at sites of inflammation. We find a localized increase in the expression of the rat interstitial collagenase (MMP-13; collagenase-3) gene from fibroblastic cells directly adjacent to macrophages within silicotic rat lung granulomas. Conditioned medium from macrophages isolated from silicotic rat lungs was found to induce rat lung fibroblast interstitial collagenase gene expression. Conditioned medium from primary rat lung macrophages or J774 monocytic cells activated by particulates in vitro also induced interstitial collagenase gene expression. Tumor necrosis factor-α (TNF-α) alone did not induce interstitial collagenase expression in rat lung fibroblasts but did in rat skin fibroblasts, revealing tissue specificity in the regulation of this gene. The activity of the conditioned medium was found to be dependent on the combined effects of TNF-α and 12-lipoxygenase-derived arachidonic acid metabolites. The fibroblast response to this conditioned medium was dependent on de novo protein synthesis and involved the induction of nuclear activator protein-1 activity. These data reveal a novel requirement for macrophage-derived 12-lipoxygenase metabolites in lung fibroblast MMP induction and provide a mechanism for the induction of resident cell MMP gene expression during inflammatory lung processes.

Muscle-specific mutations accumulate with aging in critical human mtDNA control sites for replication

The recently discovered aging-dependent large accumulation of point mutations in the human fibroblast mtDNA control region raised the question of their occurrence in postmitotic tissues. In the present work, analysis of biopsied or autopsied human skeletal muscle revealed the absence or only minimal presence of those mutations. By contrast, surprisingly, most of 26 individuals 53 to 92 years old, without a known history of neuromuscular disease, exhibited at mtDNA replication control sites in muscle an accumulation of two new point mutations, i.e., A189G and T408A, which were absent or marginally present in 19 individuals younger than 34 years. These two mutations were not found in fibroblasts from 22 subjects 64 to 101 years of age (T408A), or were present only in three subjects in very low amounts (A189G). Furthermore, in several older individuals exhibiting an accumulation in muscle of one or both of these mutations, they were nearly absent in other tissues, whereas the most frequent fibroblast-specific mutation (T414G) was present in skin, but not in muscle. Among eight additional individuals exhibiting partial denervation of their biopsied muscle, four subjects >80 years old had accumulated the two muscle-specific point mutations, which were, conversely, present at only very low levels in four subjects ≤40 years old. The striking tissue specificity of the muscle mtDNA mutations detected here and their mapping at critical sites for mtDNA replication strongly point to the involvement of a specific mutagenic machinery and to the functional relevance of these mutations.

Transposable elements as sources of variation in animals and plants

A tremendous wealth of data is accumulating on the variety and distribution of transposable elements (TEs) in natural populations. There is little doubt that TEs provide new genetic variation on a scale, and with a degree of sophistication, previously unimagined. There are many examples of mutations and other types of genetic variation associated with the activity of mobile elements. Mutant phenotypes range from subtle changes in tissue specificity to dramatic alterations in the development and organization of tissues and organs. Such changes can occur because of insertions in coding regions, but the more sophisticated TE-mediated changes are more often the result of insertions into 5′ flanking regions and introns. Here, TE-induced variation is viewed from three evolutionary perspectives that are not mutually exclusive. First, variation resulting from the intrinsic parasitic nature of TE activity is examined. Second, we describe possible coadaptations between elements and their hosts that appear to have evolved because of selection to reduce the deleterious effects of new insertions on host fitness. Finally, some possible cases are explored in which the capacity of TEs to generate variation has been exploited by their hosts. The number of well documented cases in which element sequences appear to confer useful traits on the host, although small, is growing rapidly.

p53 expression is required for thymocyte apoptosis induced by adenosine deaminase deficiency.

Adenosine deaminase (ADA, EC 3.5.4.4) is a ubiquitous enzyme in the purine catabolic pathway. In contrast to the widespread tissue distribution of this enzyme, inherited ADA deficiency in human results in a tissue-specific severe combined immunodeficiency. To explain the molecular basis for this remarkable tissue specificity, we have used a genetic approach to study ADA deficiency. We demonstrate that ADA deficiency causes depletion of CD8low transitional and CD4+CD8+ double-positive thymocytes by an apoptotic mechanism. This effect is mediated by a p53-dependent pathway, since p53-deficient mice are resistant to the apoptosis induced by ADA deficiency. DNA damage, known to be caused by the abnormal accumulation of dATP in ADA deficiency, is therefore responsible for the ablation of T-cell development and for the immunodeficiency. The two thymocyte subsets most susceptible to apoptosis induced by ADA deficiency are also the two thymocyte subsets with the lowest levels of bcl-2 expression. We show that thymocytes from transgenic mice that overexpress bcl-2 in the thymus are rescued from apoptosis induced by ADA deficiency. Thus, the tissue specificity of the pathological effects of ADA deficiency is due to the low bcl-2 expression in CD8low transitional and CD4+CD8+ double-positive thymocytes.

Origins of the specificity of tissue-type plasminogen activator.

The role of subsite interactions in defining the stringent substrate specificity of tissue-type plasminogen activator (t-PA) has been examined by using an fd phage library that displayed random hexapeptide sequences and contained 2 x 10(8) independent recombinants. Forty-four individual hexapeptides were isolated and identified as improved substrates for t-PA. A peptide containing one of the selected amino acid sequences was cleaved by t-PA 5300 times more efficiently than a peptide that contained the primary sequence of the actual cleavage site in plasminogen. These results suggest that small peptides can mimic determinants that mediate specific proteolysis, emphasize the importance of subsite interactions in determining protease specificity, and have important implications for the evolution of protease cascades.

Tissue factor- and factor X-dependent activation of protease-activated receptor 2 by factor VIIa

Protease-activated receptor 2 (PAR2) is expressed by vascular endothelial cells and other cells in which its function and physiological activator(s) are unknown. Unlike PAR1, PAR3, and PAR4, PAR2 is not activatable by thrombin. Coagulation factors VIIa (FVIIa) and Xa (FXa) are proteases that act upstream of thrombin in the coagulation cascade and require cofactors to interact with their substrates. These proteases elicit cellular responses, but their receptor(s) have not been identified. We asked whether FVIIa and FXa might activate PARs if presented by their cofactors. Co-expression of tissue factor (TF), the cellular cofactor for FVIIa, together with PAR1, PAR2, PAR3, or PAR4 conferred TF-dependent FVIIa activation of PAR2 and, to lesser degree, PAR1. Responses to FXa were also observed but were independent of exogenous cofactor. The TF/FVIIa complex converts the inactive zymogen Factor X (FX) to FXa. Strikingly, when FX was present, low picomolar concentrations of FVIIa caused robust signaling in cells expressing TF and PAR2. Responses in keratinocytes and cytokine-treated endothelial cells suggested that PAR2 may be activated directly by TF/FVIIa and indirectly by TF/FVIIa-generated FXa at naturally occurring expression levels of TF and PAR2. These results suggest that PAR2, although not activatable by thrombin, may nonetheless function as a sensor for coagulation proteases and contribute to endothelial activation in the setting of injury and inflammation. More generally, these findings highlight the potential importance of cofactors in regulating PAR function and specificity.

Identification of surface residues mediating tissue factor binding and catalytic function of the serine protease factor VIIa

Factor VIIa (VIIa), the serine protease that initiates the coagulation pathways, is catalytically activated upon binding to its cell surface receptor and cofactor tissue factor (TF). This study provides a comprehensive analysis of the functional surface of VIIa by alanine scanning mutagenesis of 112 residues. Residue side chains were defined which contribute to TF binding and factor X hydrolysis. Energetically important binding contacts at the interface with TF were identified in the first epidermal growth factor domain of VIIa (Gln-64, Ile-69, Phe-71, Arg-79) and in the protease domain (Arg-277, Met-306, Asp-309). The observed energetic defects are in good agreement with the corresponding residues in TF, suggesting that the VIIa light chain plays a prominent role in high affinity binding of cofactor. Mutation of protease domain interface residues indicated that TF allosterically influences the active site of VIIa. Stabilization of a labile zymogen to enzyme transition could explain the activating effect of TF on VIIa catalytic function. Residues important for factor X hydrolysis were found in three regions of the protease domain: (i) specificity determinants in the catalytic cleft and adjacent loops, (ii) an exosite near the TF binding site, and (iii) a large electronegative exosite which is in a position analogous to the basic exosite I of thrombin. TF regions involved in factor X activation are positioned on the same face of the TF·VIIa complex as the two exosites identified on the protease domain surface, providing evidence for an extended interaction of TF·VIIa with macromolecular substrate.

Mechanistic coupling of protease signaling and initiation of coagulation by tissue factor

The crucial role of cell signaling in hemostasis is clearly established by the action of the downstream coagulation protease thrombin that cleaves platelet-expressed G-protein-coupled protease activated receptors (PARs). Certain PARs are cleaved by the upstream coagulation proteases factor Xa (Xa) and the tissue factor (TF)–factor VIIa (VIIa) complex, but these enzymes are required at high nonphysiological concentrations and show limited recognition specificity for the scissile bond of target PARs. However, defining a physiological mechanism of PAR activation by upstream proteases is highly relevant because of the potent anti-inflammatory in vivo effects of inhibitors of the TF initiation complex. Activation of substrate factor X (X) by the TF–VIIa complex is here shown to produce enhanced cell signaling in comparison to the TF–VIIa complex alone, free Xa, or Xa that is generated in situ by the intrinsic activation complex. Macromolecular assembly of X into a ternary complex of TF–VIIa–X is required for proteolytic conversion to Xa, and product Xa remains transiently associated in a TF–VIIa–Xa complex. By trapping this complex with a unique inhibitor that preserves Xa activity, we directly show that Xa in this ternary complex efficiently activates PAR-1 and -2. These experiments support the concept that proinflammatory upstream coagulation protease signaling is mechanistically coupled and thus an integrated part of the TF–VIIa-initiated coagulation pathway, rather than a late event during excessive activation of coagulation and systemic generation of proteolytic activity.

Retrotransposons of rice involved in mutations induced by tissue culture.

Five retrotransposon families of rice (Tos1-Tos5) have been reported previously. Here we report 15 new retrotransposon families of rice (Tos6-Tos20). In contrast to yeast and Drosophila retrotransposons, all of the rice retrotransposons examined appear inactive (or almost inactive) under normal growth conditions. Three of the rice retrotransposons (Tos10, Tos17, and Tos19) are activated under tissue culture conditions. The most active one, Tos17, was studied in detail. The copy number of Tos17 increased with prolonged culture period. In all of the plants regenerated from tissue cultures, including transgenic plants, 5 to 30 transposed Tos17 copies were detected. The transcript of Tos17 was only detected under tissue culture conditions, indicating that the transposition of Tos17 is mainly regulated at the transcriptional level. To examine the target-site specificity of Tos17 transposition, sequences flanking transposed Tos17 copies were analyzed. At least four out of eight target sites examined are coding regions. Other target sites may also be in genes because two out of four were transcribed. The regenerated plants with Tos17-insertions in the phytochrome A gene and the S-receptor kinase-related gene were identified. These results indicate that activation of Tos17 is an important cause of tissue culture-induced mutations. Tissue culture-induced activation of Tos17 may be a useful tool for insertional mutagenesis and functional analysis of genes.

Regulatory region in choline acetyltransferase gene directs developmental and tissue-specific expression in transgenic mice.

Acetylcholine, one of the main neurotransmitters in the nervous system, is synthesized by the enzyme choline acetyltransferase (ChAT; acetyl-CoA:choline O-acetyltransferase, EC 2.3.1.6). The molecular mechanisms controlling the establishment, maintenance, and plasticity of the cholinergic phenotype in vivo are largely unknown. A previous report showed that a 3800-bp, but not a 1450-bp, 5' flanking segment from the rat ChAT gene promoter directed cell type-specific expression of a reporter gene in cholinergic cells in vitro. Now we have characterized a distal regulatory region of the ChAT gene that confers cholinergic specificity on a heterologous downstream promoter in a cholinergic cell line and in transgenic mice. A 2342-bp segment from the 5' flanking region of the ChAT gene behaved as an enhancer in cholinergic cells but as a repressor in noncholinergic cells in an orientation-independent manner. Combined with a heterologous basal promoter, this fragment targeted transgene expression to several cholinergic regions of the central nervous system of transgenic mice, including basal forebrain, cortex, pons, and spinal cord. In eight independent transgenic lines, the pattern of transgene expression paralleled qualitatively and quantitatively that displayed by endogenous ChAT mRNA in various regions of the rat central nervous system. In the lumbar enlargement of the spinal cord, 85-90% of the transgene expression was targeted to the ventral part of the cord, where cholinergic alpha-motor neurons are located. Transgene expression in the spinal cord was developmentally regulated and responded to nerve injury in a similar way as the endogenous ChAT gene, indicating that the 2342-bp regulatory sequence contains elements controlling the plasticity of the cholinergic phenotype in developing and injured neurons.