Molecular analysis of the SCARECROW gene in maize reveals a common basis for radial patterning in diverse meristems.

Maize and Arabidopsis root apical meristems differ in several aspects of their radial organization and ontogeny. Despite the large evolutionary distance and differences in root radial patterning, analysis of the putative maize ortholog of the Arabidopsis patterning gene SCARECROW (SCR) revealed expression localized to the endodermis, which is similar to its expression in Arabidopsis. Expression in maize extends through the quiescent center, a population of mitotically inactive cells formerly thought to be undifferentiated and to lack radial pattern information. Zea mays SCARECROW (ZmSCR), the putative maize SCR ortholog, was used as a molecular marker to investigate radial patterning during regeneration of the root tip after either whole or partial excision. Analysis of the dynamic expression pattern of ZmSCR as well as other markers indicates the involvement of positional information as a primary determinant in regeneration of the root radial pattern.

The maize caffeic acid O-methyltransferase gene promoter is active in transgenic tobacco and maize plant tissues.

The pattern of expression directed by the promoter of the maize caffeic acid O-methyltransferase (COMT) gene was studied by histochemical and fluorometric beta-glucuronidase (GUS) analysis in transgenic maize and tobacco plants. The COMT promoter directs GUS expression to the xylem and the other tissues undergoing lignification, and it responds to wounding and to elicitors. In transgenic maize plants, expression of GUS corresponds to the pattern of expression of the endogenous COMT gene as determined by northern analysis and in situ hybridization. The pattern in transgenic tobacco plants clearly shows that the maize promoter sequence is recognized by tobacco transcriptional factors, in spite of the anatomical differences and the evolutionary distance between these two species. The results suggest that the most significant promoter signals that induce the specific expression of the lignin COMT are conserved in different species.

A negatively acting DNA sequence element mediates phytochrome-directed repression of phyA gene transcription.

Phytochrome represses transcription of its own phyA genes within 5 min of light-triggered conversion to its active Pfr form. We have utilized microprojectile mediated gene transfer into etiolated rice seedlings to delineate sequence elements in the oat phyA3 promoter responsible for this regulation. Linker-scan mutagenesis of this promoter has identified two positive elements which together are necessary for maximal transcription in the absence of Pfr. These elements are designated PE1, centered at position -357 bp, and PE3, centered at position -96 bp. Sequence mutagenesis immediately downstream of PE3 results in maximal transcription in the presence of high Pfr levels, indicating that Pfr represses phyA3 transcription through a negatively acting sequence element. This element, designated RE1, with the sequence CATGGGCGCGG, encompasses a motif that is highly conserved in all monocot phyA promoters thus far characterized. DNase I protection analysis indicates that oat nuclear extracts contain multiple factors that bind to an array of sequence motifs, including PE1 and part of PE3, within 400 bp upstream of the oat phyA3 transcription start site. This DNA-binding pattern is not altered by Pfr. Weak binding to part of the RE1 motif is evident but also with no difference between high and low Pfr levels. We conclude that the signal transduction chain that mediates Pfr-directed repression of phyA3 transcription terminates with a negatively acting transcription factor that binds to the sequence element RE1.

A trans-acting factor that binds to a GT-motif in a phytochrome gene promoter.

The regulatory photoreceptor, phytochrome, controls the expression of numerous genes, including its own phyA genes, which are transcriptionally repressed in response to light. Functional analysis of a rice phyA gene promoter, by means of microprojectile-mediated gene transfer, indicates that a GT motif, GCGGTAATT, closely related to elements in the promoters of a number of other light-regulated genes, is critical for expression. Partial complementary DNA clones have been obtained for a rice nuclear protein, designated GT-2, that binds in a highly sequence-specific fashion to this motif. Mutational analysis shows that the paired G's are most crucial to binding. GT-2 has domains related to certain other transcription factors. Northern blot analysis shows that GT-2 messenger RNA levels decline in white light although red and far red light pulses are ineffective.

cis-acting elements involved in photoregulation of an oat phytochrome promoter in rice.

Phytochrome negatively regulates the transcription of its own phyA genes. High levels of Pfr, the active, far-red-light absorbing form of phytochrome, repress phyA transcription; low Pfr levels result in derepression. We have utilized microprojectile-mediated gene transfer to identify regions of an oat phyA3 gene involved in this autoregulation. Chimeric constructs containing various deletion and sequence substitution mutants of the oat phyA3 gene fused to a chloramphenicol acetyltransferase reporter (phyA3/CAT) have been introduced into etiolated rice seedlings by particle bombardment. Low Pfr concentrations induce high phyA3/CAT expression, whereas high Pfr represses activity to near basal levels. Removal of phyA3 sequences 3' to the transcription start site reduces expression about fivefold, suggesting that intron 1 of the phyA3 gene may be required for high activity. The degree of high-Pfr-imposed repression is unaffected by any of a series of deletions or sequence substitutions in the phyA3 promoter, thus providing no evidence of any Pfr-activated negative elements. In contrast, 5' and internal deletions identify a minimum of three major positive promoter elements, designated PE1 [-381 base pairs (bp) to -348 bp], PE2 (-635 bp to -489 bp), and PE3 (-110 bp to -76 bp) that are necessary for high-level expression in low-Pfr cells. The data indicate that PE1 and PE2 are functionally redundant, but that PE3 is required in conjunction with either PE1 or PE2 for activity. PE3 contains a sequence element that is highly conserved between monocot phyA promoters, indicative of a critical role in phyA expression.

Photoregulation of a phytochrome gene promoter from oat transferred into rice by particle bombardment.

The regulatory photoreceptor phytochrome controls the transcription of its own phy genes in a negative feedback fashion. We have exploited microprojectile-mediated gene transfer to develop a rapid transient expression assay system for the study of DNA sequences involved in the phytochrome-regulated expression of these genes. The 5'-flanking sequence and part of the structural region of an oat phy gene have been fused to a reporter coding sequence (chloramphenicol acetyltransferase, CAT) and introduced into intact darkgrown seedlings by using high-velocity microprojectiles. Expression is assayable in less than 24 hr from bombardment. The introduced oat phy-CAT fusion gene is expressed and down-regulated by white light in barley, rice, and oat, whereas no expression is detected in three dicots tested, tobacco, cucumber, and Arabidopsis thaliana. In bombarded rice shoots, red/far-red light-reversible repression of expression of the heterologous oat phy-CAT gene shows that it is regulated by phytochrome in a manner parallel to that of the endogenous rice phy genes. These data indicate that the transduction pathway components and promoter sequences involved in autoregulation of phy expression have been evolutionarily conserved between oat and rice. The experiments show the feasibility of using high-velocity microprojectile-mediated gene transfer for the rapid analysis of light-controlled monocot gene promoters in monocot tissues that until now have been recalcitrant to such studies.

Regulatory elements within the agropine synthase promoter of T-DNA.

DNA sequences involved in the expression of the agropine synthase gene (ags) of T-DNA were identified by analysis of transcriptional activity of promoter mutants in crown gall tumors of sunflower. Precise quantification of activity was achieved using a homologous reference gene as an internal standard. Analysis of 5'-deletion mutants demonstrated the requirement of 314 base pairs of upstream DNA sequences for optimal activity. Five regions involved in transcriptional regulation were identified in the 5'-flanking sequences between positions -74 and -314. Four of these regions make a positive contribution to promoter activity, and the fifth exerts a negative influence. The TATA motif (-26 to -33) and the TATA proximal domain (-74 to -105), which contains two sequences similar to the mammalian CCAAT box, are the major determinants of promoter activity. The two TATA distal domains A and B are separated by a negative element (-166 to -205) which may attenuate promoter strength by distancing the TATA distal domain B (-206 to -314 base pairs) from downstream components of the promoter. The TATA distal domain B contains the a/b repeat first described in the nopaline synthase (nos) promoter and was unable to support transcription in the absence of elements within the TATA proximal domain.

An enhancer-like element present in the promoter of a T-DNA gene from the Ti plasmid of Agrobacterium tumefaciens.

The promoter of the 780 gene of T-right [Thomashow, M., Nutter, R., Montoya, A., Gordon, M. & Nester, E. (1980) Cell 19, 729-739] from Agrobacterium tumefaciens Ti plasmid (pTi15955) was shown to contain an upstream cis-acting element (activator) having enhancer-like properties. To characterize the properties of this promoter element, it was placed in both polarities, upstream and downstream of a Delta-37 deletion mutant of the 780 gene. The Delta-37 deletion contains the entire 780 gene with the 5' flanking sequences deleted upstream of TATA to -37. The effect of the activator on in vivo transcriptional activity was assessed by S1 nuclease mapping utilizing a homologous reference gene as an internal standard. Transcript levels from sunflower crown gall tumors were between 127% and 90% of the wild-type 780 gene depending on the polarity of the activator element when placed directly upstream to the 780 gene Delta-37 promoter. Repositioning the activator element 613 base pairs further upstream increased transcription by 2-fold regardless of polarity. However, the activator element did not promote transcription when placed in either polarity approximately 200 base pairs downstream of the poly(A) addition site. Upstream promoter fragments (TATA-distal) from the octopine synthase (ocs) and agropine synthase (ags) genes were also tested for activation of the Delta-37 construction. The ocs sequences activated transcription of the Delta-37 deletion to 14% of wild-type levels when placed upstream in the B (reverse) orientation. All other constructions with the ocs and ags sequences showed no enhancement of promoter activity.

Functional domains of a T-DNA promoter active in crown gall tumors.

Promoter domains required for transcriptional expression of the 780 gene of T-right (pTi15955) were identified by deletion mutagenesis. Accurate quantitation of transcriptional activity of a series of 5' and internal deletion mutants was achieved by using a double gene vector containing a reference 780 gene as an internal standard. Results of the 5' deletions delineated an activator element located between -440 and -229 base pairs (bp) from the start of transcription. Removal of this region resulted in a 100-fold decrease in promoter activity. Two relatively small internal deletion/substitution mutations at positions -74 to -76 and -98 to -112 reduced promoter activity to 38 and 42%, respectively. In most cases large-scale internal deletions (38 to 151 bp) occurring in various locations from positions -12 to -348 bp caused a significant loss in major promoter activity. However, three internal deletions starting at position -37 and extending upstream as far as -153 bp either had little effect on transcriptional activity or resulted in increased activity. Removal of the TATA motif drastically reduced promoter activity to less than 0.1% of the wild type. A minor start of transcription was detected 60 bases upstream from the major transcriptional start site. This minor promoter shares the same activator element as the major promoter for full activity. Deletion and insertion mutations downstream of the minor promoter TATA demonstrated the role of the TATA box in positioning the start of transcription.

Intracranial abscesses secondary to ear and paranasal sinuses infections.

The death rate of brain abscesses in a recently reported series is high, ranging from 36% to 50% of all cases. This paper reports experiences with ten cases of intracranial abscesses secondary to ear and sinus infections. Six of these abscesses are secondary to otitic infections with three of them located in the cerebellum. Two of the cerebellar abscesses are surgically drained through the temporal bone by the otologic surgeon, with close neurosurgical cooperation. Computerized axial tomography has revolutionized the treatment of intracranial abscesses optimizing the timing for medical and surgical management.