Stress induction and developmental regulation of a rice chitinase promoter in transgenic tobacco

A 1.5 kb promoter fragment from the rice (Oryza sativa L.) RCH10 gene, which encodes a basic endochitinase inducible by wounding and fungal elicitor, was translationally fused to the β-glucuronidase (GUS) reporter gene and transferred to tobacco by Agrobacterium tumefaciens-mediated leaf disc transformation. Wounding of leaves induced GUS activity from low basal levels, and addition of fungal elicitor to the wounded tissue caused a further marked activation of the gene fusion. During vegetative development high levels of GUS activity were observed in roots and moderate levels in stems. Histochemical analysis indicated that the promoter was active in vascular and epidermal tissue, and the root apical tip. In flowers, high levels of GUS activity were observed in stigmas, ovaries and pollen-containing anthers, but only low levels in sepals and petals. The promoter 5′-deleted to ?160 exhibited the same patterns of expression in floral organs, and was also strongly induced by wounding and elicitor, but GUS activity was markedly reduced in vegetative organs. More detailed 5′ deletions showed that a cis-element required for floral expression was located between ?160 and ?74, and a cis element sufficient for stress induction was located 3′ of ?74. This proximal region 3′ of ?74 was also sufficient for expression in transfected rice protoplasts derived from suspension cultured cells. These data indicate that the complex developmental and environmental regulation of RCH10 promoter activity involves several distinct cis-elements for vegetative expression, floral expression and stress induction, and that signal pathways for wound and elicitor induction are conserved between monocotyledonous and dicotyledonous plants.     

Regulatory elements in the promoter of the vitelline membrane gene VM32E of Drosophila melanogaster direct gene expression in distinct domains of the follicular epithelium

The Drosophila vitelline membrane protein gene VM32E is expressed according to a precise temporal and spatial program in the follicle cells. Results from germ line transformation experiments using different fragments of the ?465/?39 VM32E region fused to the hsp/lacZ reporter gene revealed that the region ?348/?39 is sufficient to confer the wild-type expression pattern. Within this segment, distinct cis-regulatory elements control VM32E expression in ventral and dorsal follicle cells. The region between ?135/?113 is essential for expression of the VM32E gene in the ventral columnar follicle cells. Expression in the dorsal domain requires the two regions ?348/?254 and ?118/?39. Furthermore, the region ?253/?119 appears to contain a negative element that represses gene activity in anterior centripetal cells. We suggest that the expression of the VM32E gene throughout the follicular epithelium is controlled by specific cis-regulatory elements acting in distinct spatial domains and following a precise developmental program.     

Expression of a heat-inducible gene in transfected mosquito cells

The evolutionary conservation of the heat shock response suggests that plasmids containing promoters from Drosophila heat shock protein (hsp) genes will be useful in the development of gene transfer procedures for cell lines representing a variety of insect species. Conditions for induction of endogenous hsp genes and for expression of the chloramphenicol acetyltransferase (CAT) gene regulated by the Drosophila hsp 70 promoter were examined in Aedes albopictus (mosquito) cells. Five hsps, ranging in size from 27,000 to 90,000 D, were induced in A. albopictus cells during incubation at 41°C in medium containing [³⁵S]methionine. Relative synthesis of these proteins at 37 and 41°C indicated that Aedes hsp 66 is homologous to Drosophila hsp 70. Detection of CAT activity in transfected mosquito cells was enhanced 10-fold under heat shock conditions (6 h, 41°C) based on maximal expression of hsp 66, relative to conditions defined for expression of hsp 70 in Drosophila cells. Analysis of the endogenous heat shock response may be essential to the optimal use of plasmids containing the Drosophila hsp 70 promoter with other insect cell types.     

Expression of endogenous and microinjected hsp 30 genes in early Xenopus laevis embryos

In the present study, we have examined the regulation of expression of a newly isolated member of the hsp 30 gene family, hsp 30C. Using RT-PCR, we found that this gene was first heat-inducible at the tailbud stage of development. We also examined the expression of two microinjected modified hsp 30C gene constructs in Xenopus embryos. One of the constructs had 404 bp of hsp 30C 5′-flanking region, whereas the other had 3.6 kb. Both gene constructs had 1 kb of 3′-flanking region. RT-PCR assays were employed to detect the expression of these microinjected genes. The presence of extensive 5′- and 3′-flanking regions of the hsp 30C gene did not confer proper developmental regulation, since heat-inducible expression of both of the microinjected constructs was detectable at the midblastula stage. The premature expression of the microinjected hsp 30 gene was not a result of high plasmid copy number or the presence of plasmid DNA sequences. These results suggest that the microinjected genes contain all the cis-acting DNA sequences required for correct heat-inducible regulation but do not contain the elements required for the proper regulation of hsp 30 gene expression during development. It is possible that regulatory elements controlling the developmental expression of the hsp30 genes may reside upstream or downstream of the entire cluster. © 1993Wiley-Liss, Inc.